Novel fusions and method for detecting same

ABSTRACT

It is intended to reveal a polynucleotide serving as a novel causative gene of a cancer and, on the basis of this finding, to provide a method for detecting the polynucleotide or a polypeptide encoded thereby, a kit and a primer set for the detection, a method for screening for a substance that inhibits the polypeptide, and a pharmaceutical composition for the treatment of a cancer, containing the inhibiting substance. The detection method of the present invention detects a BRAF fusion protein or a fusion gene encoding the fusion protein, or a PXN or GMDS fusion protein or a fusion gene encoding the fusion protein in a digestive organ-derived sample obtained from a subject.

TECHNICAL FIELD

The present invention relates to a novel fusion protein containing aBRAF kinase region or a fusion gene encoding the fusion protein, and amethod for detecting the same.

The present invention also relates to a novel fusion protein containingat least a portion of PXN or GMDS or a fusion gene encoding the fusionprotein, and a method for detecting the same.

BACKGROUND ART

As a result of chromosomal translocation, a fusion gene is produced byfusing originally separate genes. It has heretofore been known that:fusion genes containing a portion of a kinase gene as a constituentoften play an essential role in carcinogenesis, as with BCR-ABL1 fusionsin chronic myelogenous leukemia, EML4-ALK fusions in lung cancer, andROS1 fusions in various cancers including lung cancer; and drugsinhibiting their functions serve as very effective anticancer agents(Non Patent Literature 1, Patent Literature 1, and Patent Literature 2).

For example, the emergence of a tyrosine kinase inhibitor crizotinib orerlotinib has motivated clinical findings on the relation of therapeuticeffects on cancers to molecular diagnosis. The concept prevails thattherapeutic drugs are administered after stratification of patients bymolecular diagnostic screening of indicated patients.

BRAF (V-Raf murine sarcoma viral oncogene homolog B1) isserine/threonine kinase belonging to the Raf kinase family and isactivated by binding to Ras-GTP (Non Patent Literature 2).

A plurality of cancers caused by BRAF fusion genes have heretofore beenknown. For example, KIAA1549-BRAF in brain tumor, SLC45A3-BRAF inprostate cancer, and AGTRAP-BRAF in stomach cancer are known (Non PatentLiteratures 3 to 5). Fusions resulting from the rearrangement of theBRAF gene and a partner gene are constantly in a phosphorylated state oftheir kinase domains and continue to send signals to the MAP kinase/ERKpathway or the like, thereby leading to the malignant transformation ofcells.

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Patent No. 4303303-   Patent Literature 2: WO2011/162295

Non Patent Literature

-   Non Patent Literature 1: Lugo, T G et al., Science. 247, 1079-1082    (1990).-   Non Patent Literature 2: Stephens R M., Mol. Cell. Biol. 12,    3733-3742 (1992).-   Non Patent Literature 3: Mark, W, ASCO Educational Book. e436-e440    (2014).-   Non Patent Literature 4: Palanisamy, N., Nat. Med., 793-799 (2010).-   Non Patent Literature 5: Mcmahon, M., Nat. Med., 749-750 (2010).

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide, on the basis ofrevealed fusions (fusion proteins and fusion genes) serving as novelcausative factors of cancers, a method for detecting a fusion protein ora fusion gene encoding the fusion protein, a method for diagnosing acancer by use of the detection method, a method for determining anapplicable subject to a pharmaceutical composition for the treatment ofa cancer, a kit and a primer set for the detection method, a method forscreening for a substance that inhibits the activity and/or expressionof a polypeptide which is the fusion protein, a pharmaceuticalcomposition for the treatment of a cancer, containing the inhibitingsubstance, and a method for treating a cancer, comprising administeringthe pharmaceutical composition for the treatment of a cancer.

Solution to Problem

The present inventors have isolated and identified a novel fusion geneof a portion of PXN gene and a portion of kinase BRAF gene fused witheach other, and a novel fusion gene of a portion of GMDS gene and aportion of kinase BRAF gene fused with each other, from samples obtainedfrom colorectal cancer patients (Examples 1 to 3) and found that thefusion genes are present in samples from colorectal cancer patients(Examples 4 and 5).

On the basis of these findings, the present inventors provide methodsfor detecting BRAF fusion proteins or fusion genes encoding the fusionproteins, provide kits and primer sets therefor, enable determination ofa cancer patient to be treated with a BRAF-inhibiting substance bydetecting the fusion protein or the fusion gene encoding the fusionprotein, and provide a method for treating a cancer, comprising the stepof administering a BRAF-inhibiting substance to the cancer patient.

On the basis of these findings, the present inventors also provide amethod for detecting a PXN or GMDS fusion protein or a fusion geneencoding the fusion protein, provide a kit and a primer set therefor,enable determination of a cancer patient to be treated with a PXN- orGMDS-inhibiting substance by detecting the fusion protein or the fusiongene encoding the fusion protein, and provide a method for treating acancer, comprising the step of administering a PXN- or GMDS-inhibitingsubstance to the cancer patient.

The present invention relates to the following aspects:

[1] A method for detecting a BRAF fusion protein or a fusion geneencoding the fusion protein in a sample obtained from a subject.[2] The detection method according to [1], wherein the detection methodcomprises the step of detecting the cleavage of the BRAF protein or thecleavage of a gene encoding the BRAF protein.[3] The detection method according to [1], wherein the detection methodcomprises the step of detecting the presence of a fusion proteinconstituted from the BRAF protein with a partner protein differenttherefrom, or the presence of a fusion gene encoding the fusion protein.[4] The detection method according to any of [1] to [3], wherein thefusion protein is a fusion protein of the BRAF protein with PXN or GMDSprotein.[5] The detection method according to any of [1] to [4], wherein thefusion protein is a polypeptide selected from the group consisting ofthe following polypeptides (a) to (d):

-   -   (a) a polypeptide consisting of the amino acid sequence        represented by SEQ ID NO: 2 (PXN-BRAF) or SEQ ID NO: 4        (GMDS-BRAF);    -   (b) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;    -   (c) a polypeptide comprising an amino acid sequence with 80% or        higher identity to the amino acid sequence represented by SEQ ID        NO: 2 or SEQ ID NO: 4, and having tumorigenicity; and    -   (d) a polypeptide comprising an amino acid sequence derived from        the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID        NO: 4 by the deletion, substitution, and/or insertion of one or        several amino acids, and having tumorigenicity.        [6] The detection method according to any of [1] to [5], wherein        the BRAF fusion gene is a polynucleotide encoding a polypeptide        according to [5].        [7] The detection method according to any of [1] to [6], wherein        the fusion gene is DNA or mRNA.        [8] The detection method according to any of [1] to [7], wherein        the sample is a digestive organ-derived sample.        [9] The detection method according to [8], wherein the digestive        organ-derived sample is a gastrointestinal tract-derived sample.        [10] The detection method according to [8], wherein the        digestive organ-derived sample is a lower gastrointestinal        tract-derived sample.        [11] The detection method according to [8], wherein the        digestive organ-derived sample is a large intestine-derived        sample.        [12] A kit for the detection of a BRAF fusion gene, comprising a        first probe capable of specifically recognizing a 5′-terminal        genomic region of the BRAF gene, and a second probe capable of        specifically recognizing a 3′-terminal genomic region of the        BRAF gene.        [13] A kit for the detection of a BRAF fusion gene, comprising a        first probe capable of specifically recognizing a 5′-terminal        genomic region of a partner gene constituting the BRAF fusion        gene together with the BRAF gene, and a second probe capable of        specifically recognizing a 3′-terminal genomic region of the        BRAF gene.        [14] A kit for the detection of a BRAF fusion gene, comprising        sense and antisense primers designed to be capable of        specifically amplifying a 5′-terminal region of a polynucleotide        encoding the BRAF protein, and sense and antisense primers        designed to be capable of specifically amplifying a 3′-terminal        region of the polynucleotide.        [15] A kit for the detection of a PXN-BRAF or GMDS-BRAF fusion        gene, comprising sense and antisense primers designed to be        capable of specifically amplifying a polynucleotide encoding a        polypeptide which is a fusion protein of the PXN or GMDS protein        with the BRAF protein.        [16] A kit for the detection of a PXN-BRAF or GMDS-BRAF fusion        gene, comprising sense and antisense primers designed to be        capable of specifically amplifying a polynucleotide encoding a        polypeptide selected from the group consisting of the following        polypeptides (a) to (d):        (a) a polypeptide consisting of the amino acid sequence        represented by SEQ ID NO: 2 or SEQ ID NO: 4;        (b) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;        (c) a polypeptide comprising an amino acid sequence with 80% or        higher identity to the amino acid sequence represented by SEQ ID        NO: 2 or SEQ ID NO: 4, and having tumorigenicity; and        (d) a polypeptide comprising an amino acid sequence derived from        the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID        NO: 4 by the deletion, substitution, and/or insertion of one or        several amino acids, and having tumorigenicity.        [17] A kit for the detection of a BRAF fusion protein,        comprising an anti-BRAF antibody capable of specifically        recognizing a N-terminal region of the BRAF protein, and an        anti-BRAF antibody capable of specifically recognizing a        C-terminal region of the BRAF protein.        [18] A kit for the detection of a BRAF fusion protein,        comprising an antibody specifically binding to a polypeptide in        a N-terminal region of a partner protein constituting the BRAF        fusion protein together with the BRAF protein, and an antibody        specifically binding to a polypeptide in a C-terminal region of        the BRAF protein.        [19] The kit according to [18], wherein the partner protein is        PXN or GMDS protein.        [20] A primer set for detecting a fusion gene of PXN or GMDS        gene with BRAF gene, comprising an antisense primer designed        from a polynucleotide moiety encoding the BRAF protein, and a        sense primer designed from a polynucleotide moiety encoding the        PXN or GMDS protein, wherein the antisense primer consists of a        nucleic acid molecule which anneals under stringent conditions        to a polynucleotide according to [16], and the sense primer        consists of a nucleic acid molecule which anneals under        stringent conditions to a complementary strand of the        polynucleotide according to [16].        [21] A primer set for detecting a fusion gene of PXN or GMDS        gene with BRAF gene, comprising an antisense primer consisting        of a nucleic acid molecule which anneals under stringent        conditions to a polynucleotide consisting of the nucleotide        sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, and a        sense primer consisting of a nucleic acid molecule which anneals        under stringent conditions to a complementary strand of the        polynucleotide.        [22] A primer set comprising a sense primer and an antisense        primer selected from the group consisting of the following sense        and antisense primers (a) and (b):        (a) a sense primer consisting of an oligonucleotide of at least        16 consecutive nucleotides arbitrarily selected from nucleotide        positions 1 to 962 of SEQ ID NO: 1, and an antisense primer        consisting of an oligonucleotide complementary to an        oligonucleotide of at least 16 consecutive nucleotides        arbitrarily selected from nucleotide positions 963 to 2067 of        SEQ ID NO: 1; and        (b) a sense primer consisting of an oligonucleotide of at least        16 consecutive nucleotides arbitrarily selected from nucleotide        positions 1 to 372 of SEQ ID NO: 3, and an antisense primer        consisting of an oligonucleotide complementary to an        oligonucleotide of at least 16 consecutive nucleotides        arbitrarily selected from nucleotide positions 373 to 1651 of        SEQ ID NO: 3.        [23] A method for screening for a substance that inhibits the        activity and/or expression of a polypeptide according to [5],        comprising the steps of:        (1) contacting the polypeptide or a cell expressing the        polypeptide with a test substance;        (2) analyzing whether or not to inhibit the activity and/or        expression of the polypeptide; and        (3) selecting the substance that inhibits the activity and/or        expression of the polypeptide.        [24] The screening method according to [23], wherein the        substance that inhibits the activity and/or expression of the        polypeptide is a therapeutic agent for a BRAF fusion-positive        cancer.        [25] The screening method according to [24], wherein the cancer        is digestive organ cancer.        [26] The screening method according to [25], wherein the cancer        is gastrointestinal cancer.        [27] The screening method according to [25], wherein the cancer        is lower gastrointestinal cancer.        [28] The screening method according to [25], wherein the cancer        is colorectal cancer.        [29] A pharmaceutical composition for the treatment of a BRAF        fusion-positive cancer, comprising a substance that inhibits the        activity and/or expression of a BRAF fusion protein.        [30] The pharmaceutical composition according to [29], wherein        the substance that inhibits the activity and/or expression of a        BRAF fusion protein is a kinase inhibitor.        [31] The pharmaceutical composition according to [29] or [30],        wherein the BRAF fusion protein is a polypeptide according to        [5].        [32] The pharmaceutical composition according to any of [29] to        [31], wherein the cancer is digestive organ cancer.        [33] The pharmaceutical composition according to [32], wherein        the cancer is gastrointestinal cancer.        [34] The pharmaceutical composition according to [32], wherein        the cancer is lower gastrointestinal cancer.        [35] The pharmaceutical composition according to [32], wherein        the cancer is colorectal cancer.        [36] A BRAF fusion protein.        [37] A fusion protein of PXN or GMDS with BRAF.        [38] The fusion protein according to [36], wherein the fusion        protein is a polypeptide selected from the group consisting of        the following polypeptides (a) to (d):        (a) a polypeptide consisting of the amino acid sequence        represented by SEQ ID NO: 2 or SEQ ID NO: 4;        (b) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;        (c) a polypeptide comprising an amino acid sequence with 80% or        higher identity to the amino acid sequence represented by SEQ ID        NO: 2 or SEQ ID NO: 4, and having tumorigenicity; and        (d) a polypeptide comprising an amino acid sequence derived from        the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID        NO: 4 by the deletion, substitution, and/or insertion of one or        several amino acids, and having tumorigenicity.        [39] A polynucleotide encoding a fusion protein according to any        of [36] to [38].        [40] A vector comprising a polynucleotide according to [39].        [41] A cell transformed with a vector according to [40].        [42] A method for detecting a PXN or GMDS fusion protein or a        fusion gene encoding the fusion protein in a sample obtained        from a subject.        [43] The detection method according to [42], wherein the        detection method comprises the step of detecting the cleavage of        the PXN or GMDS protein or the cleavage of a gene encoding the        PXN or GMDS protein.        [44] The detection method according to [42], wherein the        detection method comprises the step of detecting the presence of        a fusion protein constituted from the PXN or GMDS protein with a        partner protein different therefrom or the presence of a fusion        gene encoding the fusion protein.        [45] The detection method according to any of [42] to [44],        wherein the fusion protein is a fusion protein of the PXN or        GMDS protein with BRAF protein.        [46] The detection method according to any of [42] to [45],        wherein the fusion protein is a polypeptide selected from the        group consisting of the following polypeptides (a) to (d):        (a) a polypeptide consisting of the amino acid sequence        represented by SEQ ID NO: 2 or SEQ ID NO: 4;        (b) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;        (c) a polypeptide comprising an amino acid sequence with 80% or        higher identity to the amino acid sequence represented by SEQ ID        NO: 2 or SEQ ID NO: 4, and having tumorigenicity; and        (d) a polypeptide comprising an amino acid sequence derived from        the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID        NO: 4 by the deletion, substitution, and/or insertion of one or        several amino acids, and having tumorigenicity.        [47] The detection method according to any of [42] to [46],        wherein the PXN or GMDS fusion gene is a polynucleotide encoding        a polypeptide according to [46].        [48] The detection method according to any of [42] to [47],        wherein the fusion gene is DNA or mRNA.        [49] The detection method according to any of [42] to [48],        wherein the sample is a digestive organ-derived sample.        [50] The detection method according to [49], wherein the        digestive organ-derived sample is a gastrointestinal        tract-derived sample.        [51] The detection method according to [49], wherein the        digestive organ-derived sample is a lower gastrointestinal        tract-derived sample.        [52] The detection method according to [49], wherein the        digestive organ-derived sample is a large intestine-derived        sample.        [53] A kit for the detection of a PXN or GMDS fusion gene,        comprising a first probe capable of specifically recognizing a        5′-terminal genomic region of the PXN or GMDS gene, and a second        probe capable of specifically recognizing a 3′-terminal genomic        region of the PXN or GMDS gene.        [54] A kit for the detection of a PXN or GMDS fusion gene,        comprising a first probe capable of specifically recognizing a        3′-terminal genomic region of a partner gene constituting the        PXN or GMDS fusion gene together with the PXN or GMDS gene, and        a second probe capable of specifically recognizing a 5′-terminal        genomic region of the PXN or GMDS gene.        [55] A kit for the detection of a PXN or GMDS fusion gene,        comprising sense and antisense primers designed to be capable of        specifically amplifying a 5′-terminal region of a polynucleotide        encoding the PXN or GMDS protein, and sense and antisense        primers designed to be capable of specifically amplifying a        3′-terminal region of the polynucleotide.        [56] A kit for the detection of a PXN-BRAF or GMDS-BRAF fusion        gene, comprising sense and antisense primers designed to be        capable of specifically amplifying a polynucleotide encoding a        polypeptide which is a fusion protein of the PXN or GMDS protein        with the BRAF protein.        [57] A kit for the detection of a PXN-BRAF or GMDS-BRAF fusion        gene, comprising sense and antisense primers designed to be        capable of specifically amplifying a polynucleotide encoding a        polypeptide selected from the group consisting of the following        polypeptides (a) to (d):        (a) a polypeptide consisting of the amino acid sequence        represented by SEQ ID NO: 2 or SEQ ID NO: 4;        (b) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;        (c) a polypeptide comprising an amino acid sequence with 80% or        higher identity to the amino acid sequence represented by SEQ ID        NO: 2 or SEQ ID NO: 4, and having tumorigenicity; and        (d) a polypeptide comprising an amino acid sequence derived from        the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID        NO: 4 by the deletion, substitution, and/or insertion of one or        several amino acids, and having tumorigenicity.        [58] A kit for the detection of a PXN or GMDS fusion protein,        comprising an anti-PXN or anti-GMDS antibody capable of        specifically recognizing a N-terminal region of the PXN or GMDS        protein, and an anti-PXN or anti-GMDS antibody capable of        specifically recognizing a C-terminal region of the PXN or GMDS        protein.        [59] A kit for the detection of a PXN or GMDS fusion protein,        comprising an antibody specifically binding to a polypeptide in        a C-terminal region of a partner protein constituting the PXN or        GMDS fusion protein together with the PXN or GMDS protein, and        an antibody specifically binding to a polypeptide in a        N-terminal region of the PXN or GMDS protein.        [60] The kit according to [59], wherein the partner protein is        BRAF protein.        [61] A primer set for detecting a fusion gene of BRAF gene with        PXN or GMDS gene, comprising an antisense primer designed from a        polynucleotide moiety encoding the BRAF protein, and a sense        primer designed from a polynucleotide moiety encoding the PXN or        GMDS protein, wherein the antisense primer consists of a nucleic        acid molecule which anneals under stringent conditions to a        polynucleotide according to [57], and the sense primer consists        of a nucleic acid molecule which anneals under stringent        conditions to a complementary strand of the polynucleotide        according to [57].        [62] A primer set for detecting a fusion gene of PXN or GMDS        gene with BRAF gene, comprising an antisense primer consisting        of a nucleic acid molecule which anneals under stringent        conditions to a polynucleotide consisting of the nucleotide        sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, and a        sense primer consisting of a nucleic acid molecule which anneals        under stringent conditions to a complementary strand of the        polynucleotide.        [63] A primer set comprising a sense primer and an antisense        primer selected from the group consisting of the following sense        and antisense primers (a) and (b):        (a) a sense primer consisting of an oligonucleotide of at least        16 consecutive nucleotides arbitrarily selected from nucleotide        positions 1 to 962 of SEQ ID NO: 1, and an antisense primer        consisting of an oligonucleotide complementary to an        oligonucleotide of at least 16 consecutive nucleotides        arbitrarily selected from nucleotide positions 963 to 2067 of        SEQ ID NO: 1; and        (b) a sense primer consisting of an oligonucleotide of at least        16 consecutive nucleotides arbitrarily selected from nucleotide        positions 1 to 372 of SEQ ID NO: 3, and an antisense primer        consisting of an oligonucleotide complementary to an        oligonucleotide of at least 16 consecutive nucleotides        arbitrarily selected from nucleotide positions 373 to 1651 of        SEQ ID NO: 3.        [64] A method for screening for a substance that inhibits the        activity and/or expression of a polypeptide according to [46],        comprising the steps of:        (1) contacting the polypeptide or a cell expressing the        polypeptide with a test substance;        (2) analyzing whether or not to inhibit the activity and/or        expression of the polypeptide; and        (3) selecting the substance that inhibits the activity and/or        expression of the polypeptide.        [65] The screening method according to [64], wherein the        substance that inhibits the activity and/or expression of the        polypeptide is a therapeutic agent for a PXN or GMDS        fusion-positive cancer.        [66] The screening method according to [65], wherein the cancer        is digestive organ cancer.        [67] The screening method according to [66], wherein the cancer        is gastrointestinal cancer.        [68] The screening method according to [66], wherein the cancer        is lower gastrointestinal cancer.        [69] The screening method according to [66], wherein the cancer        is colorectal cancer.        [70] A pharmaceutical composition for the treatment of a PXN or        GMDS fusion-positive cancer, comprising a substance that        inhibits the activity and/or expression of a PXN or GMDS fusion        protein.        [71] The pharmaceutical composition according to [70], wherein        the substance that inhibits the activity and/or expression of a        PXN or GMDS fusion protein is a kinase inhibitor.        [72] The pharmaceutical composition according to [70] or [71],        wherein the PXN or GMDS fusion protein is a polypeptide        according to [46].        [73] The pharmaceutical composition according to any of [70] to        [72], wherein the cancer is digestive organ cancer.        [74] The pharmaceutical composition according to [73], wherein        the cancer is gastrointestinal cancer.        [75] The pharmaceutical composition according to [73], wherein        the cancer is lower gastrointestinal cancer.        [76] The pharmaceutical composition according to [73], wherein        the cancer is colorectal cancer.        [77] A PXN or GMDS fusion protein.        [78] A fusion protein of PXN or GMDS with BRAF.        [79] The fusion protein according to [77], wherein the fusion        protein is a polypeptide selected from the group consisting of        the following polypeptides (a) to (d):        (a) a polypeptide consisting of the amino acid sequence        represented by SEQ ID NO: 2 or SEQ ID NO: 4;        (b) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;        (c) a polypeptide comprising an amino acid sequence with 80% or        higher identity to the amino acid sequence represented by SEQ ID        NO: 2 or SEQ ID NO: 4, and having tumorigenicity; and        (d) a polypeptide comprising an amino acid sequence derived from        the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID        NO: 4 by the deletion, substitution, and/or insertion of one or        several amino acids, and having tumorigenicity.        [80] A polynucleotide encoding a fusion protein according to any        of [77] to [79].        [81] A vector comprising a polynucleotide according to [80].        [82] A cell transformed with a vector according to [81].        [83] A method for treating a BRAF fusion-positive cancer,        wherein a substance that inhibits the activity and/or expression        of a BRAF fusion protein is a kinase inhibitor.        [84] Use of a substance that inhibits the activity and/or        expression of a BRAF fusion protein in the production of a        pharmaceutical composition for the treatment of a BRAF        fusion-positive cancer.        [85] A method for treating a PXN or GMDS fusion-positive cancer,        wherein a substance that inhibits the activity and/or expression        of a PXN or GMDS fusion protein is a kinase inhibitor.        [86] Use of a substance that inhibits the activity and/or        expression of a PXN or GMDS fusion protein in the production of        a pharmaceutical composition for the treatment of a PXN or GMDS        fusion-positive cancer.

Advantageous Effects of Invention

The detection method of the present invention can be used as a methodfor detecting a BRAF fusion-positive cancer (particularly, digestiveorgan cancer). According to the detection method of the presentinvention, a BRAF fusion-positive cancer in a subject can be diagnosed,and further, whether or not to be an applicable subject to aBRAF-inhibiting substance can be determined. The kit and the primer setfor detection of the present invention can be used in the detectionmethod of the present invention. Furthermore, the inhibiting substancescreening method of the present invention can screen for a substanceeffective for the treatment of a patient with the fusion-positivecancer. The substance obtained by the screening method can be used as anactive ingredient in a pharmaceutical composition for the treatment of aBRAF fusion-positive cancer and can also be used in the treatment of aBRAF fusion-positive cancer.

The detection method of the present invention can be used as a methodfor detecting a PXN or GMDS fusion-positive cancer (particularly,digestive organ cancer). According to the detection method of thepresent invention, a PXN or GMDS fusion-positive cancer in a subject canbe diagnosed, and further, whether or not to be an applicable subject toa PXN- or GMDS-inhibiting substance can be determined. The kit and theprimer set for detection of the present invention can be used in thedetection method of the present invention. Furthermore, the inhibitingsubstance screening method of the present invention can screen for asubstance effective for the treatment of a patient with thefusion-positive cancer. The substance obtained by the screening methodcan be used as an active ingredient in a pharmaceutical composition forthe treatment of a PXN or GMDS fusion-positive cancer and can also beused in the treatment of a PXN or GMDS fusion-positive cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a microscopic photograph, instead of a drawing, showing thestate of NIH3T3 fibroblasts transfected with a fusion gene PXN-BRAF andcultured for 7 days.

FIG. 2 is a microscopic photograph, instead of a drawing, showing thestate of NIH3T3 fibroblasts transfected with a fusion gene GMDS-BRAF andcultured for 7 days.

FIG. 3 is graph showing time-dependent change in tumor size from 1 to 8days after inoculation in nude mice subcutaneously inoculated with the3T3 fibroblasts transfected with the fusion gene PXN-BRAF or GMDS-BRAF.

FIG. 4 is a graph showing the sensitivity of Ba/F3 cells expressing aPXN-BRAF or GMDS-BRAF fusion polypeptide to a BRAF inhibitor(trametinib).

FIG. 5 is a photograph, instead of a drawing, showing results oftreating Ba/F3 cells expressing a PXN-BRAF or GMDS-BRAF fusionpolypeptide with a BRAF inhibitor and then carrying out the Westernblotting of each cultured cell-derived extract.

DESCRIPTION OF EMBODIMENTS Definition, Etc.

<Fusion Point>

In the present specification, the phrase “fusion point in a BRAF fusiongene” means a position at which a polynucleotide derived from the BRAFgene and a polynucleotide derived from a partner gene that constitutesthe fusion gene together with the BRAF gene are joined with each otherin the BRAF fusion gene.

In the present specification, the phrase “fusion point in a PXN or GMDSfusion gene” means a position at which a polynucleotide derived from thePXN or GMDS gene and a polynucleotide derived from a partner gene thatconstitutes the fusion gene together with the PXN or GMDS gene arejoined with each other in the PXN or GMDS fusion gene.

When the BRAF fusion gene or the PXN or GMDS fusion gene is, forexample, a PXN-BRAF fusion gene shown in SEQ ID NO: 1 (PXNex6-BRAFex11),the fusion point is a position (position 962/963) at which the3′-terminal nucleotide (position 962) of the polynucleotide derived fromthe PXN gene and the 5′-terminal nucleotide (position 963) of thepolynucleotide derived from the BRAF gene are joined with each other.

When the BRAF fusion gene or the PXN or GMDS fusion gene is a GMDS-BRAFfusion gene shown in SEQ ID NO: 3 (GMDSex1-BRAFex9), the fusion point isa position (position 372/373) at which the 3′-terminal nucleotide(position 372) of the polynucleotide derived from the GMDS gene and the5′-terminal nucleotide (position 373) of the polynucleotide derived fromthe BRAF gene are joined with each other.

In the present specification, the phrase “fusion point in a BRAF fusionprotein” means a position at which a polypeptide encoded by thepolynucleotide derived from the BRAF gene and a polypeptide encoded bythe polynucleotide derived from the partner gene that constitutes thefusion gene together with the BRAF gene are joined with each other inthe BRAF fusion protein.

In the present specification, the phrase “fusion point in a PXN or GMDSfusion protein” means a position at which a polypeptide encoded by thepolynucleotide derived from the PXN or GMDS gene and a polypeptideencoded by the polynucleotide derived from the partner gene thatconstitutes the fusion gene together with the PXN or GMDS gene arejoined with each other in the PXN or GMDS fusion protein.

When the BRAF fusion protein or the PXN or GMDS fusion protein is, forexample, a PXN-BRAF fusion protein shown in SEQ ID NO: 2, the fusionpoint is a position (position 277/278) at which the C-terminal aminoacid (position 277) of the polypeptide derived from the PXN protein andthe N-terminal amino acid (position 278) of the polypeptide derived fromthe BRAF protein are joined with each other.

When the BRAF fusion protein or the PXN or GMDS fusion protein is aGMDS-BRAF fusion protein shown in SEQ ID NO: 4, the fusion point is aposition (position 34/35) at which the C-terminal amino acid (position34) of the polypeptide derived from the GMDS protein and the N-terminalamino acid (position 35) of the polypeptide derived from the BRAFprotein are joined with each other.

<Cleavage of BRAF Gene or BRAF Protein>

In the present specification, the phrase “cleavage of the BRAF gene” or“BRAF gene is cleaved” refers to a state where the continuity of theBRAF gene is lost due to the translocation or inversion, etc. of thegene, i.e., a state where the BRAF gene is separated into at least twopolynucleotides, a polynucleotide containing a BRAF kinase region andanother polynucleotide. The break point of the BRAF gene is not limitedas long as a protein encoded by at least one of the polynucleotidesresulting from the cleavage of the BRAF gene retains BRAF kinaseactivity.

Also, the phrase “cleavage of a partner gene different from the BRAFgene” or “partner gene different from the BRAF gene is cleaved” refersto a state where the continuity of the partner gene is lost due to thetranslocation or inversion, etc. of the gene, i.e., a state where thepartner gene is separated into at least two polynucleotides.

In the present specification, the phrase “cleavage of the BRAF protein”or “BRAF protein is cleaved” refers to a state where the continuity ofthe BRAF protein is lost, i.e., a state where the BRAF protein isseparated into at least two polypeptides, a polypeptide containing aBRAF kinase region and another polypeptide, on the basis of the cleavedstate of the BRAF gene as mentioned above. The break point of the BRAFprotein is not limited as long as at least one of the polypeptidesresulting from the cleavage of the BRAF protein retains BRAF kinaseactivity.

Also, the phrase “cleavage of a partner protein different from the BRAFprotein” or “partner protein different from the BRAF protein is cleaved”refers to a state where the continuity of the partner protein is lost,i.e., a state where the partner protein is separated into at least twopolypeptides, on the basis of the cleaved state of the partner gene asmentioned above.

<Cleavage of PXN or GMDS Gene or PXN or GMDS Protein>

In the present specification, the phrase “cleavage of the PXN or GMDSgene” or “PXN or GMDS gene is cleaved” refers to a state where thecontinuity of the PXN or GMDS gene is lost due to the translocation orinversion, etc. of the gene. The break point of the PXN or GMDS gene isnot limited as long as a protein encoded by a partner gene thatconstitutes the PXN or GMDS fusion gene together with the PXN or GMDSgene retains its function (e.g., kinase activity when this protein has akinase domain).

Also, the phrase “cleavage of a partner gene different from the PXN orGMDS gene” or “partner gene different from the PXN or GMDS gene iscleaved” refers to a state where the continuity of the partner gene islost due to the translocation or inversion, etc. of the gene, i.e., astate where the partner gene is separated into at least twopolynucleotides.

In the present specification, the phrase “cleavage of the PXN or GMDSprotein” or “PXN or GMDS protein is cleaved” refers to a state where thecontinuity of the PXN or GMDS protein is lost, i.e., a state where thePXN or GMDS protein is separated into at least two polypeptides, on thebasis of the cleaved state of the PXN or GMDS gene as mentioned above.The break point of the PXN or GMDS protein is not limited as long as apartner protein that constitutes the PXN or GMDS fusion protein togetherwith the PXN or GMDS protein retains its function (e.g., kinase activitywhen this protein has a kinase domain).

Also, the phrase “cleavage of a partner protein different from the PXNor GMDS protein” or “partner protein different from the PXN or GMDSprotein is cleaved” refers to a state where the continuity of thepartner protein is lost, i.e., a state where the partner protein isseparated into at least two polypeptides, on the basis of the cleavedstate of the partner gene as mentioned above.

<5′-Terminal Region/3′-Terminal Region and N-Terminal Region/C-TerminalRegion>

The 5′-terminal region refers to, in the case of a fusion gene, apolynucleotide located on the 5′-terminal side with respect to thefusion point and, in the case of a wild-type gene (gene which is not afusion gene), a polynucleotide located on the 5′-terminal side withrespect to the break point when the wild-type gene constitutes a fusiongene. The 5′-terminal region may be a region in any of genomic DNA,mRNA, and cDNA and is also referred to as, for example, a 5′-terminalgenomic region in the genomic DNA.

The 3′-terminal region refers to, in the case of a fusion gene, apolynucleotide located on the 3′-terminal side with respect to thefusion point and, in the case of a wild-type gene (gene which is not afusion gene), a polynucleotide located on the 3′-terminal side withrespect to the break point when the wild-type gene constitutes a fusiongene. The 3′-terminal region may be a region in any of genomic DNA,mRNA, and cDNA and is also referred to as, for example, a 3′-terminalgenomic region in the genomic DNA.

The N-terminal region refers to, in the case of a fusion protein, apolypeptide located on the N-terminal side with respect to the fusionpoint and, in the case of a wild-type protein (protein which is not afusion protein), a polynucleotide located on the N-terminal side withrespect to the break point when the wild-type protein constitutes afusion gene.

The C-terminal region refers to, in the case of a fusion protein, apolypeptide located on the C-terminal side with respect to the fusionpoint and, in the case of a wild-type protein (protein which is not afusion protein), a polynucleotide located on the C-terminal side withrespect to the break point when the wild-type protein constitutes afusion gene.

For example, in a PXN-BRAF fusion gene shown in SEQ ID NO: 1(PXNex6-BRAFex11), the 5′-terminal region is a polynucleotide consistingof a nucleotide sequence from positions 1 to 962, and the 3′-terminalregion is a polynucleotide consisting of a nucleotide sequence frompositions 963 to 2067. In a PXN-BRAF fusion protein shown in SEQ ID NO:2, the N-terminal region is a polypeptide (amino acid positions 1 to 277of SEQ ID NO: 2) encoded by CDS (nucleotide positions 132 to 962 of SEQID NO: 1) in the 5′-terminal region of the PXNex6-BRAFex11, and theC-terminal region is a polypeptide (amino acid positions 278 to 605 ofSEQ ID NO: 2) encoded by CDS (nucleotide positions 963 to 1949 of SEQ IDNO: 1) in the 3′-terminal region of the PXNex6-BRAFex11.

In a GMDS-BRAF fusion gene shown in SEQ ID NO: 3 (GMDSex1-BRAFex9), the5′-terminal region is a polynucleotide consisting of a nucleotidesequence from positions 1 to 372, and the 3′-terminal region is apolynucleotide consisting of a nucleotide sequence from positions 373 to1651. In a GMDS-BRAF fusion protein shown in SEQ ID NO: 4, theN-terminal region is a polypeptide (amino acid positions 1 to 34 of SEQID NO: 4) encoded by CDS (nucleotide positions 271 to 372 of SEQ ID NO:3) in the 5′-terminal region of the GMDSex1-BRAFex9, and the C-terminalregion is a polypeptide (amino acid positions 35 to 420 of SEQ ID NO: 4)encoded by CDS (nucleotide positions 373 to 1533 of SEQ ID NO: 3) in the3′-terminal region of the GMDSex1-BRAFex9.

<cDNA Reference Sequence>

In the present specification, ENST00000288602 for BRAF, ENST00000267257for PXN, and ENST00000380815 for GMDS were used as cDNA referencesequences of the genes, and ENSP00000288602 for BRAF, ENSP00000267257for PXN, and ENSP00000370194 for GMDS were used as amino acid referencesequences of the proteins.

<Stringent Conditions>

In the present specification, the term “stringent conditions” refers toconditions involving hybridization conditions of “5×SSPE, 5×Denhardt'ssolution, 0.5% SDS, 50% formamide, 200 μg/mL salmon sperm DNA, 42° C.overnight” and washing conditions of “0.5×SSC, 0.1% SDS, 42° C.”. Theterm “more stringent conditions” refers to conditions involvinghybridization conditions of “5×SSPE, 5×Denhardt's solution, 0.5% SDS,50% formamide, 200 μg/mL salmon sperm DNA, 42° C., overnight” andwashing conditions of “0.2×SSC, 0.1% SDS, 65° C.”.

<Tumorigenicity>

Whether a polypeptide “has tumorigenicity” can be confirmed by a methodknown in the art, for example, a method described in Example 4 ofWO2011/162295 or a method described in Example 6 mentioned later.Specifically, the confirmation method involves subcutaneouslyinoculating a host (3T3 fibroblasts) transfected with an expressionplasmid for the polypeptide to a nude mouse and determining the presenceor absence of tumor formation.

Sample for Use in Detection Method of Present Invention

<Target Organ>

The detection method according to the present invention can be suitablyused in the detection of a cancer developed in a target organ. The testsite (target organ) of a subject is not limited as long as the fusionaccording to the present invention is present therein. The test site ispreferably a digestive organ, more preferably the gastrointestinaltract, further preferably the gastrointestine, still further preferablythe lower gastrointestinal tract, particularly preferably the largeintestine.

The histological type of the test site is not limited as long as thedetection method according to the present invention is applicablethereto. The histological type may be a squamous epithelial tissue ormay be a glandular tissue and is preferably a squamous epithelialtissue.

<Harvest from Subject>

In the detection method according to the present invention, a harvestfrom a subject (sample separated from an organism), specifically, anarbitrary harvested body fluid (preferably blood), a resected samplefrom an affected area in a subject, a biopsy sample or a swab sample,feces, urine, a gastrointestinal lavage fluid, or the like can be usedas a sample obtained from a subject. The gastrointestinal lavage fluidmay be a lavage fluid of the whole gastrointestinal tract or may be alavage fluid of the gastrointestinal tract containing at least the testsite, for example, a lavage fluid of the lower gastrointestinal tract ora lavage fluid of the large intestine. The sample obtained from asubject is preferably a sample containing cells of the test site in thetarget organ, more preferably a resected sample or a biopsy sample fromthe test site of the subject, in consideration of detection sensitivity.

<Preparation of Harvest>

The method for detecting a BRAF fusion gene or a BRAF fusion proteinaccording to the present invention can be carried out by preparing atissue section or a cell suspension, etc. of a sample obtained from asubject and detecting the BRAF fusion gene or the BRAF fusion protein incells contained in the tissue section or the cell suspension by atechnique well known to those skilled in the art. Alternatively, alysate is prepared from the sample obtained from the subject mentionedabove, and genes or proteins contained therein are extracted. In thisextracted sample, the BRAF fusion gene or the BRAF fusion protein may bedetected by a technique well known to those skilled in the art. Thedetection of the BRAF fusion gene may be the detection of genomic DNA ofthe BRAF fusion gene or may be the detection of mRNA which is atranscript of the genomic DNA or cDNA obtained with the mRNA as atemplate.

The method for detecting a PXN or GMDS fusion gene or a PXN or GMDSfusion protein according to the present invention can be carried out bypreparing a tissue section or a cell suspension, etc. of a sampleobtained from a subject and detecting the PXN or GMDS fusion gene or thePXN or GMDS fusion protein in cells contained in the tissue section orthe cell suspension by a technique well known to those skilled in theart. Alternatively, a lysate is prepared from the sample obtained fromthe subject mentioned above, and genes or proteins contained therein areextracted. In this extracted sample, the PXN or GMDS fusion gene or thePXN or GMDS fusion protein may be detected by a technique well known tothose skilled in the art. The detection of the PXN or GMDS fusion genemay be the detection of genomic DNA of the PXN or GMDS fusion gene ormay be the detection of mRNA which is a transcript of the genomic DNA orcDNA obtained with the mRNA as a template.

Target to be Detected by Detection Method of Present Invention

The detection method of the present invention includes a method fordetecting a BRAF fusion, i.e., a method for detecting a fusion proteincontaining a BRAF kinase region (also referred to as a “BRAF fusionprotein”), or a method for detecting a fusion gene encoding the fusionprotein (also referred to as a “BRAF fusion gene”), in a sample obtainedfrom a subject.

The detection method of the present invention includes a method fordetecting a PXN or GMDS fusion, i.e., a method for detecting a PXN orGMDS fusion protein, or a method for detecting a fusion gene encodingthe fusion protein (also referred to as a “PXN or GMDS fusion gene”), ina sample obtained from a subject.

<BRAF Fusion: BRAF Fusion Protein and BRAF Fusion Gene>

The BRAF fusion according to the present invention includes a BRAFfusion protein and a BRAF fusion gene.

The BRAF fusion protein according to the present invention is a fusionpolypeptide constituted from a polypeptide derived from the BRAF proteinand a polypeptide derived from a partner protein different from the BRAFprotein. The polypeptide derived from the BRAF protein is notparticularly limited as long as the polypeptide comprises at least apolypeptide having a BRAF kinase region in the BRAF protein. Thepolypeptide derived from the partner protein different from the BRAFprotein is not particularly limited as long as the polypeptide comprisesat least a partial polypeptide of the partner protein.

The partner protein is not particularly limited as long as the BRAFfusion protein constituted by its fusion with a BRAF kinasedomain-containing portion of the BRAF protein has tumorigenicity. It ispreferred that the constituted BRAF fusion protein should constantlymaintain BRAF kinase activation and thereby have tumorigenicity.

The BRAF fusion protein may comprise a third polypeptide which isneither the polypeptide derived from the BRAF protein nor thepolypeptide derived from the partner protein different from the BRAFprotein as long as the constituted BRAF fusion protein constantlymaintains BRAF kinase activation and has tumorigenicity. The thirdpolypeptide may be positioned at the N terminus of the BRAF fusionprotein, may be positioned at the C terminus thereof, or may bepositioned between the polypeptide derived from the BRAF protein and thepolypeptide derived from the partner protein different from the BRAFprotein.

The BRAF fusion protein is particularly preferably a fusion proteinhaving PXN or GMDS protein as the partner protein. Specifically, theBRAF fusion protein is preferably a fusion protein of the PXN or GMDSprotein with the BRAF protein (hereinafter, also referred to as aPXN-BRAF fusion protein or a GMDS-BRAF fusion protein, a PXN-BRAF orGMDS-BRAF fusion protein, or a PXN- or GMDS-BRAF fusion protein)constituted from a polypeptide derived from the BRAF protein, comprisingat least a polypeptide having a BRAF kinase region, and a polypeptidederived from the PXN or GMDS protein, comprising at least a partialpolypeptide of the PXN or GMDS protein.

<PXN or GMDS Fusion: PXN or GMDS Fusion Protein and PXN or GMDS FusionGene>

The PXN or GMDS fusion according to the present invention includes a PXNor GMDS fusion protein and a PXN or GMDS fusion gene.

The PXN or GMDS fusion protein according to the present invention is afusion polypeptide constituted from a polypeptide derived from the PXNor GMDS protein and a polypeptide derived from a partner proteindifferent from the PXN or GMDS protein. The polypeptide derived from thePXN or GMDS protein is not particularly limited as long as thepolypeptide comprises at least a partial polypeptide of the PXN or GMDSprotein. The polypeptide derived from the partner protein different fromthe PXN or GMDS protein is not particularly limited as long as thepolypeptide comprises at least a partial polypeptide of the partnerprotein.

The partner protein is not particularly limited as long as the PXN orGMDS fusion protein constituted by its fusion with a portion of the PXNor GMDS protein has tumorigenicity. It is preferred that the PXN or GMDSfusion protein should constantly maintain the activation of a functionaldomain (preferably a kinase domain) carried by the partner protein andthereby have tumorigenicity.

The PXN or GMDS fusion protein may comprise a third polypeptide which isneither the polypeptide derived from the PXN or GMDS protein nor thepolypeptide derived from the partner protein different from the PXN orGMDS protein as long as the constituted PXN or GMDS fusion proteinconstantly maintains the activation of a functional domain of thepartner protein different from the PXN or GMDS protein by fusion with aportion of the PXN or GMDS protein and has tumorigenicity. The thirdpolypeptide may be positioned at the N terminus of the PXN or GMDSfusion protein, may be positioned at the C terminus thereof, or may bepositioned between the polypeptide derived from the PXN or GMDS proteinand the polypeptide derived from the partner protein different from thePXN or GMDS protein.

The PXN or GMDS fusion protein is particularly preferably a fusionprotein having BRAF protein as the partner protein. Specifically, thePXN or GMDS fusion protein is preferably a fusion protein of the PXN orGMDS protein with the BRAF protein (hereinafter, also referred to as aPXN-BRAF fusion protein or a GMDS-BRAF fusion protein, a PXN-BRAF orGMDS-BRAF fusion protein, or a PXN- or GMDS-BRAF fusion protein)constituted from a polypeptide derived from the PXN or GMDS protein,comprising at least a partial polypeptide of the PXN or GMDS protein,and at least a partial polypeptide of the BRAF protein, comprising atleast a polypeptide having a BRAF kinase region.

The “PXN- or GMDS-BRAF fusion protein” is particularly preferably any ofthe following polypeptides (a) to (d):

(a) a polypeptide consisting of the amino acid sequence represented bySEQ ID NO: 2 (PXN-BRAF) or SEQ ID NO: 4 (GMDS-BRAF);

(b) a polypeptide comprising the amino acid sequence represented by SEQID NO: 2 or SEQ ID NO: 4, and having tumorigenicity;

(c) a polypeptide comprising an amino acid sequence with 80% or higheridentity to the amino acid sequence represented by SEQ ID NO: 2 or SEQID NO: 4, and having tumorigenicity (hereinafter, referred to as ahomologous polypeptide); and

(d) a polypeptide comprising an amino acid sequence derived from theamino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 by thedeletion, substitution, and/or insertion of one or several amino acids,and having tumorigenicity (hereinafter, referred to as a functionallyequivalent variant).

The amino acid sequence represented by SEQ ID NO: 2 is a sequenceencoded by the nucleotide sequence represented by SEQ ID NO: 1,particularly, the nucleotide sequence represented by nucleotidepositions 132 to 1949 (CDS) of SEQ ID NO: 1. The nucleotide sequencerepresented by SEQ ID NO: 1 consists of a nucleotide sequence having the5′-UTR sequence of the PXN gene, start codon ATG to exon 6 of the PXNgene, exon 11 to stop codon at exon 18 of the BRAF gene, and the 3′-UTRsequence of the BRAF gene. In the nucleotide sequence represented by SEQID NO: 1, the sequence from nucleotide positions 1 to 962 is derivedfrom the PXN gene, and the sequence from nucleotide positions 963 to2067 is derived from the BRAF gene. In the present specification, thepolypeptide consisting of the amino acid sequence represented by SEQ IDNO: 2, and a polynucleotide consisting of a nucleotide sequence encodingthis polypeptide (including a polynucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 1) are collectivelyreferred to as a PXNex6-BRAFex11 fusion (or simply PXNex6-BRAFex11).

The amino acid sequence represented by SEQ ID NO: 4 is a sequenceencoded by the nucleotide sequence represented by SEQ ID NO: 3,particularly, the nucleotide sequence represented by nucleotidepositions 271 to 1533 (CDS) of SEQ ID NO: 3. The nucleotide sequencerepresented by SEQ ID NO: 3 consists of a nucleotide sequence having the5′-UTR sequence of the GMDS gene, start codon ATG to exon 1 of the GMDSgene, exon 9 to stop codon at exon 18 of the BRAF gene, and the 3′-UTRsequence of the BRAF gene. In the nucleotide sequence represented by SEQID NO: 3, the sequence from nucleotide positions 1 to 372 is derivedfrom the GMDS gene, and the sequence from nucleotide positions 373 to1651 is derived from the BRAF gene. In the present specification, thepolypeptide consisting of the amino acid sequence represented by SEQ IDNO: 4, and a polynucleotide consisting of a nucleotide sequence encodingthis polypeptide (including a polynucleotide consisting of thenucleotide sequence represented by SEQ ID NO: 3) are collectivelyreferred to as a GMDSex1-BRAFex9 fusion (or simply GMDSex1-BRAFex9).

In the “functionally equivalent variant”, the number of amino acids thatcan be substituted, deleted, and/or inserted is 1 to several, preferably1 to 10, more preferably 1 to 7, most preferably 1 to 5.

The “homologous polypeptide” is a “polypeptide comprising an amino acidsequence with 80% or higher identity to the amino acid sequencerepresented by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity”,preferably a polypeptide comprising an amino acid sequence with theidentity of preferably 90% or higher, more preferably 95% or higher,further preferably 98% or higher. The “polypeptide comprising an aminoacid sequence with 80% or higher identity to the amino acid sequencerepresented by SEQ ID NO: 2 or SEQ ID NO: 4, and having tumorigenicity”includes a polypeptide that exhibits the identity described above and apolypeptide having at least one substitution, deletion, and/or insertion(preferably substitution) (homologous polypeptide in the narrow sense)and a polypeptide having 100% identity.

In the present specification, the “identity” means a value Identityobtained using parameters provided as defaults by NEEDLE program (J MolBiol 1970; 48: 443-453) search. The parameters are as follows:

Gap penalty=10

Extend penalty=0.5

Matrix=EBLOSUM62

The BRAF fusion gene according to the present invention is apolynucleotide encoding the BRAF fusion protein. In the presentspecification, the BRAF fusion protein and the BRAF fusion gene are alsocollectively referred to as a “BRAF fusion”.

The PXN or GMDS fusion gene according to the present invention is apolynucleotide encoding the PXN or GMDS fusion protein. Specifically,the PXN fusion gene is a polynucleotide encoding the PXN fusion protein,and the GMDS fusion gene is a polynucleotide encoding the GMDS fusionprotein. In the present specification, the PXN or GMDS fusion proteinand the PXN or GMDS fusion gene are also collectively referred to as a“PXN or GMDS fusion”.

The BRAF fusion according to the present invention is preferably aPXNex6-BRAFex11 fusion variant or a GMDSex1-BRAFex9 fusion variant.Particularly, the BRAF fusion protein according to the present inventionis preferably a PXNex6-BRAFex11 fusion protein variant or aGMDSex1-BRAFex9 fusion protein variant. Also, the BRAF fusion geneaccording to the present invention is preferably a PXNex6-BRAFex11fusion gene variant or a GMDSex1-BRAFex9 fusion gene variant.

The PXN or GMDS fusion according to the present invention is preferablya PXNex6-BRAFex11 fusion variant or a GMDSex1-BRAFex9 fusion variant.Particularly, the PXN or GMDS fusion protein according to the presentinvention is preferably a PXNex6-BRAFex11 fusion protein variant or aGMDSex1-BRAFex9 fusion protein variant. Also, the PXN or GMDS fusiongene according to the present invention is preferably a PXNex6-BRAFex11fusion gene variant or a GMDSex1-BRAFex9 fusion gene variant.

Aspect of Detection Method of Present Invention (Methods for DetectingFusion Protein and Fusion Gene)

The detection method of the present invention includes a detectionmethod comprising the step of detecting the cleavage of the BRAF proteinor the cleavage of the BRAF gene encoding the BRAF protein in a sampleobtained from a subject, and a detection method comprising the step ofdetecting the presence of a fusion protein constituted from the BRAFprotein with a partner protein different from the BRAF protein, or thepresence of a fusion gene encoding the fusion protein in a sampleobtained from a subject.

The detection method of the present invention includes a detectionmethod comprising the step of detecting the cleavage of the PXN or GMDSprotein or the cleavage of the PXN or GMDS gene encoding the PXN or GMDSprotein in a sample obtained from a subject, and a detection methodcomprising the step of detecting the presence of a fusion proteinconstituted from the PXN or GMDS protein with a partner proteindifferent from the PXN or GMDS protein, or the presence of a fusion geneencoding the fusion protein in a sample obtained from a subject.

<Aspect of Detecting BRAF Fusion Gene>

Hereinafter, aspects of detecting the BRAF fusion gene will bedescribed, but are not limited to those described below.

The detection of a particular region in a gene in each aspect givenbelow may be performed using a probe or a primer designed on the basisof a nucleotide sequence analyzed in advance or may be performed bysequencing, regardless of the examples thereof.

[Aspect (1) of Detecting BRAF Fusion Gene]

<Aspect (1-a) of Detecting BRAF Fusion Gene>

The BRAF gene is cleaved into two or more polynucleotides when the BRAFfusion gene is constituted. On the basis of this event, in one aspect ofdetecting the BRAF fusion gene, the BRAF fusion gene can be detected bydetecting the state where the BRAF gene is cleaved, i.e., the continuitybetween the 5′-terminal region of the BRAF gene and the 3′-terminalregion of the BRAF gene is lost.

Specifically, the BRAF fusion gene can be detected, for example, byusing a first probe which specifically hybridizes to the 5′-terminalregion of the BRAF gene and a second probe which specifically hybridizesto the 3′-terminal region of the BRAF gene and detecting these two generegions located distant from each other on the chromosome.

The BRAF fusion gene may be detected by confirming the state where thepartner gene constituting the fusion gene by its fusion with thepolynucleotide derived from the BRAF gene is cleaved, according to themethod described above.

<Aspect (1-b) of Detecting BRAF Fusion Gene>

In an alternative aspect, the BRAF fusion gene can be detected byspecifically detecting the respective expression levels of the5′-terminal region and the 3′-terminal region of the BRAF gene anddetermining the ratio between the expression levels. Specifically, theBRAF fusion gene can be detected, for example, when the expression levelof the 5′-terminal region of the BRAF gene and the expression level ofthe 3′-terminal region of the BRAF gene are different from each other.

Alternatively, the BRAF fusion gene may be detected by confirming thisas to the partner gene, different from the BRAF gene, constituting theBRAF fusion gene together with the BRAF gene, according to the methoddescribed above.

<Aspect (1-c) of Detecting BRAF Fusion Gene>

In an alternative aspect, the process of formation of the BRAF fusiongene may involve the duplication of at least a portion of the BRAF geneor the partner gene different from the BRAF gene. In other words, theBRAF fusion gene may be constituted from a duplicated polynucleotidederived from the BRAF gene and a duplicated polynucleotide derived fromthe partner gene, different from the BRAF gene, constituting the BRAFfusion gene together with the BRAF gene. In this case, the BRAF fusiongene can be detected by detecting the duplication of the polynucleotidederived from the BRAF gene or the polynucleotide derived from thepartner gene.

[Aspect (2) of Detecting BRAF Fusion Gene]

The BRAF fusion gene is constituted by the fusion between thepolynucleotide derived from the BRAF gene and the polynucleotide derivedfrom the partner gene different from the BRAF gene. On the basis of thisevent, in one aspect of detecting the BRAF fusion gene, the BRAF fusiongene can be detected by detecting a fusion polynucleotide consecutivelycomprising at least a portion of the polynucleotide derived from theBRAF gene and at least a portion of the polynucleotide derived from thegene different from the BRAF gene in the BRAF fusion gene.

Specifically, the BRAF fusion gene can be detected, for example, byusing a first probe which specifically hybridizes to the 5′-terminalregion of the polynucleotide derived from the partner gene differentfrom the BRAF gene, and a second probe which specifically hybridizes tothe 3′-terminal region of the polynucleotide derived from the BRAF gene,and detecting these two gene regions located in proximity on thechromosome. When the partner gene different from the BRAF gene is PXN orGMDS gene, i.e., the BRAF fusion gene is a PXN- or GMDS-BRAF fusiongene, the first probe can employ a probe which specifically hybridizesto the 5′-terminal region of the polynucleotide derived from the PXN orGMDS gene.

[Aspect (3) of Detecting BRAF Fusion Gene]

The BRAF fusion gene is constituted by the fusion at the fusion pointbetween the polynucleotide derived from the BRAF gene and thepolynucleotide derived from the partner gene different from the BRAFgene. On the basis of this event, in one aspect of detecting the BRAFfusion gene, the BRAF fusion gene can be detected by detecting a fusionpolynucleotide consecutively comprising at least a portion of thepolynucleotide derived from the BRAF gene and at least a portion of thepolynucleotide derived from the partner gene different from the BRAFgene in the BRAF fusion gene, and containing the fusion point.

Specifically, the BRAF fusion gene can be detected, for example, byperforming PCR reaction using a first primer which specifically annealsto the 5′-terminal region of the polynucleotide derived from the partnergene different from the BRAF gene, and a second primer whichspecifically anneals to the 3′-terminal region of the polynucleotidederived from the BRAF gene, and confirming that a predetermined PCRproduct that indicates the presence of the fusion point is obtained.

<Aspect of Detecting BRAF Fusion Protein>

Hereinafter, aspects of detecting the BRAF fusion protein will bedescribed, but are not limited to those described below.

[Aspect (1) of Detecting BRAF Fusion Protein]

<Aspect (1-a) of Detecting BRAF Fusion Protein>

The BRAF protein encoded by the BRAF gene is also cleaved when the BRAFfusion gene is constituted. On the basis of this event, in an aspect ofdetecting the BRAF fusion protein, the BRAF fusion protein can bedetected by detecting the state where the BRAF protein is cleaved, i.e.,the continuity between the N-terminal region and the C-terminal regionof the BRAF protein is lost.

Specifically, the BRAF fusion protein can be detected, for example, byusing a first antibody specifically binding to the N-terminal region ofthe BRAF protein, and a second antibody specifically binding to theC-terminal region of the BRAF protein, and confirming that these tworegions are absent in the same protein.

Alternatively, the BRAF fusion protein may be detected by confirming thestate where the partner protein, different from the BRAF protein,constituting the fusion protein together with the BRAF protein iscleaved, according to the method described above.

<Aspect (1-b) of Detecting BRAF Fusion Protein>

In an alternative aspect, the BRAF fusion protein can be detected byspecifically detecting the respective expression levels of theN-terminal region and the C-terminal region of the BRAF protein anddetermining the ratio between the expression levels. Specifically, theBRAF fusion protein can be detected, for example, by using, as an index,the difference between the expression level of the N-terminal region ofthe BRAF protein and the expression level of the C-terminal region ofthe BRAF protein.

Alternatively, the BRAF fusion protein may be detected by confirmingthis as to the partner protein, different from the BRAF protein,constituting the BRAF fusion protein together with the BRAF protein,according to the method described above.

[Aspect (2) of Detecting BRAF Fusion Protein]

The BRAF fusion protein is constituted by the fusion between thepolypeptide derived from the BRAF protein and the polypeptide derivedfrom the partner protein different from the BRAF protein. On the basisof this event, in one aspect of detecting the BRAF fusion protein, theBRAF fusion protein can be detected by detecting a fusion polypeptideconsecutively comprising at least a portion of the polypeptide derivedfrom the BRAF protein and at least a portion of the polypeptide derivedfrom the partner protein in the BRAF fusion protein.

Specifically, the BRAF fusion protein can be detected, for example, byusing a first antibody specifically binding to the N-terminal region ofthe partner protein different from the BRAF protein, and a secondantibody specifically binding to the C-terminal region of the BRAFprotein, and confirming that these two regions are present in the sameprotein.

[Aspect (3) of Detecting BRAF Fusion Protein]

The BRAF fusion protein is constituted by the fusion at the fusion pointbetween the polypeptide derived from the BRAF protein and thepolypeptide derived from the partner protein different from the BRAFprotein. On the basis of this event, in one aspect of detecting the BRAFfusion protein, the BRAF fusion protein can be detected by detecting afusion polypeptide consecutively comprising at least a portion of thepolypeptide derived from the BRAF protein and at least a portion of thepolypeptide derived from the partner protein in the BRAF fusion protein,and containing the fusion point.

Specifically, the BRAF fusion protein can be detected, for example, byimmunoassay using an antibody specifically recognizing a polypeptidecontaining the fusion point of the BRAF fusion protein.

[Aspect (4) of Detecting BRAF Fusion Protein]

In one aspect of detecting the BRAF fusion protein, the BRAF fusionprotein can be detected by using the activity of the BRAF fusion proteinas an index.

Specifically, the BRAF fusion protein can be detected, for example, byusing a substance having inhibitory activity against wild-type BRAFprotein to inhibit the activity of the wild-type BRAF protein, thenmeasuring the kinase activity of the BRAF protein, and using, as anindex, higher activity than that in the absence of the BRAF fusionprotein (in the presence of only the wild-type BRAF protein). For themeasurement of the kinase activity of the BRAF protein, a method wellknown to those skilled in the art can be appropriately selected, and,for example, the phosphorylated state of a molecule that undergoesphosphorylation by BRAF may be detected.

The detection of the BRAF fusion protein may be performed by using, asan index, the presence of a full-length polypeptide constituting theBRAF fusion protein or the presence of a polypeptide constituting aportion of the BRAF fusion protein, and is not limited as long as thepresence of the BRAF fusion protein can be confirmed.

<Aspect of Detecting PXN or GMDS Fusion Gene>

Hereinafter, aspects of detecting the PXN or GMDS fusion gene will bedescribed, but are not limited to those described below.

The detection of a particular region in a gene in each aspect givenbelow may be performed using a probe or a primer designed on the basisof a nucleotide sequence analyzed in advance or may be performed bysequencing, regardless of the examples thereof.

[Aspect (1) of Detecting PXN or GMDS Fusion Gene]

<Aspect (1-a) of Detecting PXN or GMDS Fusion Gene>

The PXN or GMDS gene is cleaved into two or more polynucleotides whenthe PXN or GMDS fusion gene is constituted. On the basis of this event,in one aspect of detecting the PXN or GMDS fusion gene, the PXN or GMDSfusion gene can be detected by detecting the state where the PXN or GMDSgene is cleaved, i.e., the continuity between the 5′-terminal region ofthe PXN or GMDS gene and the 3′-terminal region of the PXN or GMDS geneis lost.

Specifically, the PXN or GMDS fusion gene can be detected, for example,by using a first probe which specifically hybridizes to the 5′-terminalregion of the PXN or GMDS gene and a second probe which specificallyhybridizes to the 3′-terminal region of the PXN or GMDS gene anddetecting these two gene regions located distant from each other on thechromosome.

The PXN or GMDS fusion gene may be detected by confirming the statewhere the partner gene constituting the fusion gene by its fusion withthe polynucleotide derived from the PXN or GMDS gene is cleaved,according to the method described above.

<Aspect (1-b) of Detecting PXN or GMDS Fusion Gene>

In an alternative aspect, the PXN or GMDS fusion gene can be detected byspecifically detecting the respective expression levels of the5′-terminal region and the 3′-terminal region of the PXN or GMDS geneand determining the ratio between the expression levels. Specifically,the PXN or GMDS fusion gene can be detected, for example, when theexpression level of the 5′-terminal region of the PXN or GMDS gene andthe expression level of the 3′-terminal region of the PXN or GMDS geneare different from each other.

Alternatively, the PXN or GMDS fusion gene may be detected by confirmingthis as to the partner gene, different from the PXN or GMDS gene,constituting the PXN or GMDS fusion gene together with the PXN or GMDSgene, according to the method described above.

<Aspect (1-c) of Detecting PXN or GMDS Fusion Gene>

The process of formation of the PXN or GMDS fusion gene may involve theduplication of at least a portion of the PXN or GMDS gene or the partnergene different from the PXN or GMDS gene. In other words, the PXN orGMDS fusion gene may be constituted from a duplicated polynucleotidederived from the PXN or GMDS gene and a duplicated polynucleotidederived from the partner gene, different from the PXN or GMDS gene,constituting the PXN or GMDS fusion gene together with the PXN or GMDSgene. In this case, in an alternative aspect, the PXN or GMDS fusiongene can be detected by detecting the duplication of the polynucleotidederived from the PXN or GMDS gene or the polynucleotide derived from thepartner gene.

[Aspect (2) of Detecting PXN or GMDS Fusion Gene]

The PXN or GMDS fusion gene is constituted by the fusion between thepolynucleotide derived from the PXN or GMDS gene and the polynucleotidederived from the partner gene different from the PXN or GMDS gene. Onthe basis of this event, in one aspect of detecting the PXN or GMDSfusion gene, the PXN or GMDS fusion gene can be detected by detecting afusion polynucleotide consecutively comprising at least a portion of thepolynucleotide derived from the PXN or GMDS gene and at least a portionof the polynucleotide derived from the gene different from the PXN orGMDS gene in the PXN or GMDS fusion gene.

Specifically, the PXN or GMDS fusion gene can be detected, for example,by using a first probe which specifically hybridizes to the 5′-terminalregion of the polynucleotide derived from the PXN or GMDS gene, and asecond probe which specifically hybridizes to the 3′-terminal region ofthe polynucleotide derived from the partner gene different from the PXNor GMDS gene, and detecting these two gene regions located in proximityon the chromosome. When the partner gene different from the PXN or GMDSgene is BRAF gene, i.e., the PXN or GMDS fusion gene is a PXN- orGMDS-BRAF fusion gene, the second probe can employ a probe whichspecifically hybridizes to the 3′-terminal region of the polynucleotidederived from the BRAF gene.

[Aspect (3) of Detecting PXN or GMDS Fusion Gene]

The PXN or GMDS fusion gene is constituted by the fusion at the fusionpoint between the polynucleotide derived from the PXN or GMDS gene andthe polynucleotide derived from the partner gene different from the PXNor GMDS gene. On the basis of this event, in one aspect of detecting thePXN or GMDS fusion gene, the PXN or GMDS fusion gene can be detected bydetecting a fusion polynucleotide consecutively comprising at least aportion of the polynucleotide derived from the PXN or GMDS gene and atleast a portion of the polynucleotide derived from the partner genedifferent from the PXN or GMDS gene in the PXN or GMDS fusion gene, andcontaining the fusion point.

Specifically, the PXN or GMDS fusion gene can be detected, for example,by performing PCR reaction using a first primer which specificallyanneals to the 5′-terminal region of the polynucleotide derived from thePXN or GMDS gene, and a second primer which specifically anneals to the3′-terminal region of the polynucleotide derived from the partner genedifferent from the PXN or GMDS gene, and confirming that a predeterminedPCR product that indicates the presence of the fusion point is obtained.

<Aspect of Detecting PXN or GMDS Fusion Protein>

Hereinafter, aspects of detecting the PXN or GMDS fusion protein will bedescribed, but are not limited to those described below.

[Aspect (1) of Detecting PXN or GMDS Fusion Protein]

<Aspect (1-a) of Detecting PXN or GMDS Fusion Protein>

The PXN or GMDS protein encoded by the PXN or GMDS gene is also cleavedwhen the PXN or GMDS fusion gene is constituted. On the basis of thisevent, in an aspect of detecting the PXN or GMDS fusion protein, the PXNor GMDS fusion protein can be detected by detecting the state where thePXN or GMDS protein is cleaved, i.e., the continuity between theN-terminal region and the C-terminal region of the PXN or GMDS proteinis lost.

Specifically, the PXN or GMDS fusion protein can be detected, forexample, by using a first antibody specifically binding to theN-terminal region of the PXN or GMDS protein, and a second antibodyspecifically binding to the C-terminal region of the PXN or GMDSprotein, and confirming that these two regions are absent in the sameprotein.

Alternatively, the PXN or GMDS fusion protein may be detected byconfirming the state where the partner protein, different from the PXNor GMDS protein, constituting the fusion protein together with the PXNor GMDS protein is cleaved, according to the method described above.

<Aspect (1-b) of Detecting PXN or GMDS Fusion Protein>

In an alternative aspect, the PXN or GMDS fusion protein can be detectedby specifically detecting the respective expression levels of theN-terminal region and the C-terminal region of the PXN or GMDS proteinand determining the ratio between the expression levels. Specifically,the PXN or GMDS fusion protein can be detected, for example, by using,as an index, the difference between the expression level of theN-terminal region of the PXN or GMDS protein and the expression level ofthe C-terminal region of the PXN or GMDS protein.

Alternatively, the PXN or GMDS fusion protein may be detected byconfirming this as to the partner protein, different from the PXN orGMDS protein, constituting the PXN or GMDS fusion protein together withthe PXN or GMDS protein, according to the method described above.

[Aspect (2) of Detecting PXN or GMDS Fusion Protein]

The PXN or GMDS fusion protein is constituted by the fusion between thepolypeptide derived from the PXN or GMDS protein and the polypeptidederived from the partner protein different from the PXN or GMDS protein.On the basis of this event, in one aspect of detecting the PXN or GMDSfusion protein, the PXN or GMDS fusion protein can be detected bydetecting a fusion polypeptide consecutively comprising at least aportion of the polypeptide derived from the PXN or GMDS protein and atleast a portion of the polypeptide derived from the partner protein inthe PXN or GMDS fusion protein.

Specifically, the PXN or GMDS fusion protein can be detected, forexample, by using a first antibody specifically binding to theN-terminal region of the PXN or GMDS protein, and a second antibodyspecifically binding to the C-terminal region of the partner proteindifferent from the PXN or GMDS protein, and confirming that these tworegions are present in the same protein.

[Aspect (3) of Detecting PXN or GMDS Fusion Protein]

The PXN or GMDS fusion protein is constituted by the fusion at thefusion point between the polypeptide derived from the PXN or GMDSprotein and the polypeptide derived from the partner protein differentfrom the PXN or GMDS protein. On the basis of this event, in one aspectof detecting the PXN or GMDS fusion protein, the PXN or GMDS fusionprotein can be detected by detecting a fusion polypeptide consecutivelycomprising at least a portion of the polypeptide derived from the PXN orGMDS protein and at least a portion of the polypeptide derived from thepartner protein in the PXN or GMDS fusion protein, and containing thefusion point.

Specifically, the PXN or GMDS fusion protein can be detected, forexample, by immunoassay using an antibody specifically recognizing apolypeptide containing the fusion point of the PXN or GMDS fusionprotein.

[Aspect (4) of Detecting PXN or GMDS Fusion Protein]

In one aspect of detecting the PXN or GMDS fusion protein, the PXN orGMDS fusion protein can be detected by using the activity of the PXN orGMDS fusion protein as an index.

Specifically, when the partner protein, different from the PXN or GMDSprotein, constituting the fusion protein together with the PXN or GMDSprotein is a protein having enzymatic activity, the PXN or GMDS fusionprotein can be detected, for example, by using, as an index, higherenzymatic activity concerned than that in the absence of the PXN or GMDSfusion protein (in the presence of only the wild-type PXN or GMDSprotein). For the measurement of the enzymatic activity, a method wellknown to those skilled in the art can be appropriately selected. Whenthe partner protein is, for example, a protein having kinase activity(preferably BRAF protein), the phosphorylated state of a molecule thatundergoes phosphorylation by PXN or GMDS fusion protein may be detected.

The detection of the PXN or GMDS fusion protein may be performed byusing, as an index, the presence of a full-length polypeptideconstituting the PXN or GMDS fusion protein or the presence of apolypeptide constituting a portion of the PXN or GMDS fusion protein,and is not limited as long as the presence of the PXN or GMDS fusionprotein can be confirmed.

Technique for Use in Detection Method

Hereinafter, each step and each detection technique for the detection ofthe BRAF fusion gene (genomic DNA, mRNA, or cDNA), the detection of thePXN or GMDS fusion gene (genomic DNA, mRNA, or cDNA), the detection ofthe BRAF fusion protein, and the detection of the PXN or GMDS fusionprotein will be described in more detail, but are not limited to thosedescribed below.

In the case of extracting a gene (genomic DNA or mRNA) or a protein froma sample obtained from a subject or in the case of preparing a tissuesection or a cell suspension, etc. therefrom, a suitable technique fordetecting the BRAF fusion gene or the PXN or GMDS fusion gene, or theBRAF fusion protein or the PXN or GMDS fusion protein in the preparedsample can be appropriately selected by those skilled in the art.

<Detection of Fusion Gene>

The detection of the BRAF fusion gene or the PXN or GMDS fusion gene maybe the detection of genomic DNA of the BRAF fusion gene or the PXN orGMDS fusion gene, the detection of mRNA which is a transcript of thegenomic DNA, or the detection of cDNA obtained with the mRNA as atemplate.

Any technique well known to those skilled in the art for use in genedetection, such as a hybridization technique using a probe (nucleic acidprobe, etc.) which hybridizes to at least a portion of the BRAF fusiongene or the PXN or GMDS fusion gene, or a gene amplification techniqueusing a primer which anneals to at least a portion of the BRAF fusiongene or the PXN or GMDS fusion gene, and applied techniques based onthese techniques can be used as a technique of detecting the BRAF fusiongene (genomic DNA or mRNA) or the PXN or GMDS fusion gene (genomic DNAor mRNA) in a sample obtained from a subject.

Specifically, any technique may be used, such as PCR, LCR (ligase chainreaction), SDA (strand displacement amplification), NASBA (nucleic acidsequence-based amplification), ICAN (isothermal and chimericprimer-initiated amplification of nucleic acids), LAMP (loop-mediatedisothermal amplification), TMA (Gen-Probe's TMA system), in situhybridization, microarray, Northern hybridization, Southernhybridization, dot blot, RNA protection, DNA sequencing, or RNAsequencing.

[Detection of Genomic DNA]

An in situ hybridization technique can be suitably used in the detectionof the genomic DNA. The detection using the in situ hybridizationtechnique can be carried out according to, for example, FISH known inthe art. Alternatively, this detection can be carried out by fusionassay based on chromogenic in situ hybridization (CISH) and silver insitu hybridization (SISH) in combination. Preferably, the genomic DNAcan be detected by FISH split assay or FISH fusion assay described indetail below.

Alternatively, a DNA sequencing technique can be suitably used in thedetection of the genomic DNA. The sequencing may employ a conventionalsequencer based on the Sanger method and preferably employs anext-generation sequencer in consideration of analysis efficiency (seee.g., Metzker M L, Nat Rev Genet. 2010 January; 11 (1): 31-46). Examplesof the next-generation sequencer can include MiSeq/HiSeq from Illumina,Inc., SOLiD System from Life Technologies Corp., and 454 SequencingSystem (GS FLX+/GS Junior) from F. Hoffmann-La Roche, Ltd. In thesequencing, the efficiency of analysis can be improved by enriching aregion likely to contain the fusion gene using a sequencing capturetechnique or the like. Examples of the sequencing capture technique caninclude Roche NimbleGen from F. Hoffmann-La Roche, Ltd. and Sure Selectfrom Agilent Technologies, Inc.

Hereinafter, typical methods for detecting the genomic DNA will beillustrated, but are not limited to those described below.

<FISH Split Assay>

In the FISH split assay for the BRAF fusion gene, a fluorescentlylabeled polynucleotide covering the 5′-terminal genomic region of theBRAF gene, and a labeled (with another fluorescent dye) polynucleotidecovering the 3′-terminal genomic region of this gene are used incombination as probes for detection. A color resulting from the overlapof two signals (e.g., yellow in the case of using a red fluorescent dyeand a green fluorescent dye) is detected under normal conditions (in thecase of wild-type BRAF gene), because the two gene regions (5′-terminalregion and 3′-terminal region of each gene) are located in proximity. Onthe other hand, separate and distant signals (e.g., red and green)derived from the two types of fluorescent dyes are detected when thegene is cleaved into two regions due to translocation or inversion.Thus, the FISH split assay detects the presence of the BRAF fusion geneby detecting the 5′-terminal genomic region and the 3′-terminal genomicregion of the BRAF gene located distant from each other on thechromosome.

In the FISH split assay for the PXN or GMDS fusion gene, a fluorescentlylabeled polynucleotide covering the 5′-terminal genomic region of thePXN or GMDS gene, and a labeled (with another fluorescent dye)polynucleotide covering the 3′-terminal genomic region of this gene areused in combination as probes for detection. A color resulting from theoverlap of two signals (e.g., yellow in the case of using a redfluorescent dye and a green fluorescent dye) is detected under normalconditions (in the case of wild-type PXN or GMDS gene), because the twogene regions (5′-terminal region and 3′-terminal region of each gene)are located in proximity. On the other hand, separate and distantsignals (e.g., red and green) derived from the two types of fluorescentdyes are detected when the gene is cleaved into two regions due totranslocation or inversion. Thus, the FISH split assay detects thepresence of the PXN or GMDS fusion gene by detecting the 5′-terminalgenomic region and the 3′-terminal genomic region of the PXN or GMDSgene located distant from each other on the chromosome.

When the BRAF fusion gene or the PXN or GMDS fusion gene is a PXN- orGMDS-BRAF fusion gene, the PXN- or GMDS-BRAF fusion gene can be detectedby using a fluorescently labeled polynucleotide covering the 5′-terminalgenomic region of the PXN or GMDS gene, and a labeled (with anotherfluorescent dye) polynucleotide covering the 3′-terminal genomic regionof this gene, or a fluorescently labeled polynucleotide covering the5′-terminal genomic region of the BRAF gene, and a labeled (with anotherfluorescent dye) polynucleotide covering the 3′-terminal genomic regionof this gene, in combination as probes for detection.

<FISH Fusion Assay>

In the FISH fusion assay for the BRAF fusion gene, when the BRAF fusiongene is, for example, a PXN- or GMDS-BRAF fusion gene, a fluorescentlylabeled polynucleotide covering the 3′-terminal genomic region of theBRAF gene, and a labeled (with another fluorescent dye) polynucleotidecovering the 5′-terminal genomic region of the PXN or GMDS gene can beused in combination as probes for detection. Separate and distantsignals (e.g., red and green) derived from the two types of fluorescentdyes are detected under normal conditions (in the case of wild-type BRAFgene). On the other hand, a color resulting from the overlap of twosignals (e.g., yellow) is detected when the two gene regions are locatedin proximity due to translocation or inversion.

In the FISH fusion assay for the PXN or GMDS fusion gene, when the PXNor GMDS fusion gene is, for example, a PXN- or GMDS-BRAF fusion gene, afluorescently labeled polynucleotide covering the 3′-terminal genomicregion of the BRAF gene, and a labeled (with another fluorescent dye)polynucleotide covering the 5′-terminal genomic region of the PXN orGMDS gene can be used in combination as probes for detection. Separateand distant signals (e.g., red and green) derived from the two types offluorescent dyes are detected under normal conditions (in the case ofwild-type PXN or GMDS gene). On the other hand, a color resulting fromthe overlap of two signals (e.g., yellow) is detected when the two generegions are located in proximity due to translocation or inversion.

<Detection of Gene Duplication by Use of FISH>

In the detection of gene duplication associated with BRAF fusion geneconstruction, when the BRAF fusion gene is, for example, a PXN- orGMDS-BRAF fusion gene, a fluorescently labeled polynucleotide coveringat least a portion of the 3′-terminal genomic region of the BRAF genecan be used as a probe for detection. The BRAF fusion gene can bedetected by detecting the obtainment of a stronger signal, for example,a signal two or more times stronger, than that in the presence of onlywild-type BRAF gene.

The BRAF fusion gene may be detected by the method described above usinga probe for the detection of the 5′-terminal genomic region of thepartner gene (e.g., the PXN or GMDS gene when the BRAF fusion gene is aPXN- or GMDS-BRAF fusion gene) constituting the fusion gene by itsfusion with the polynucleotide derived from the BRAF gene.

In the detection of gene duplication associated with PXN or GMDS fusiongene construction, when the PXN or GMDS fusion gene is, for example, aPXN- or GMDS-BRAF fusion gene, a fluorescently labeled polynucleotidecovering at least a portion of the 5′-terminal genomic region of the PXNor GMDS gene can be used as a probe for detection. The PXN or GMDSfusion gene can be detected by detecting the obtainment of a strongersignal, for example, a signal two or more times stronger, than that inthe presence of only wild-type PXN or GMDS gene.

The PXN or GMDS fusion gene may be detected by the method describedabove using a probe for the detection of the 3′-terminal genomic regionof the partner gene (e.g., the BRAF gene when the PXN or GMDS fusiongene is a BRAF-PXN or -GMDS fusion gene) constituting the fusion gene byits fusion with the polynucleotide derived from the PXN or GMDS gene.

<Detection of Gene Duplication by Use of CGH Array Analysis>

The gene duplication associated with BRAF fusion gene construction orPXN or GMDS fusion gene construction can be detected by comparativegenomic hybridization (CGH) array analysis (e.g., Agilent CGH/CNV ArrayAnalysis; Agilent Technologies, Inc.).

<Detection of Gene Duplication Using Next-Generation Sequencer>

The gene duplication associated with BRAF fusion gene construction orPXN or GMDS fusion gene construction can be detected using anext-generation sequencer. Specifically, in analysis using thenext-generation sequencer, the BRAF fusion gene or the PXN or GMDSfusion gene can be detected by detecting high coverage of a geneduplication site (high redundancy of this site when the sequence of theDNA fragment to be analyzed is annotated with a reference sequence).

<Probe for Use in Detection (for Genome)>

The probe for use in hybridization for detecting the BRAF fusion gene ispreferably a probe which hybridizes under stringent conditions(preferably under more stringent conditions) to at least partialnucleotides of the BRAF fusion gene or a complementary strand thereof.

In the case of detecting, for example, genomic DNA of the BRAF fusiongene containing the fusion point, a probe comprising a nucleic acidmolecule of at least 32 consecutive bases consisting of 16 upstream and16 downstream bases flanking the fusion point of the BRAF fusion gene,or a complementary strand thereof may be used.

The probe for use in hybridization for detecting the PXN or GMDS fusiongene is preferably a probe which hybridizes under stringent conditions(preferably under more stringent conditions) to at least partialnucleotides of the PXN or GMDS fusion gene or a complementary strandthereof.

In the case of detecting, for example, genomic DNA of the PXN or GMDSfusion gene containing the fusion point, a probe comprising a nucleicacid molecule of at least 32 consecutive bases consisting of 16 upstreamand 16 downstream bases flanking the fusion point of the PXN or GMDSfusion gene, or a complementary strand thereof may be used.

When the BRAF fusion gene or the PXN or GMDS fusion gene is, forexample, a PXN- or GMDS-BRAF fusion gene, a first probe capable ofspecifically recognizing the 5′-terminal genomic region of any one ofthe BRAF gene and the PXN or GMDS gene, and a second probe capable ofspecifically recognizing the 3′-terminal genomic region of the othergene (preferably a first probe capable of specifically recognizing the3′-terminal genomic region of the BRAF gene, and a second probe capableof specifically recognizing the 5′-terminal genomic region of the PXN orGMDS gene) can be used in combination as the probes capable of using inthe FISH fusion assay.

Meanwhile, when the BRAF fusion gene or the PXN or GMDS fusion gene is,for example, a PXN- or GMDS-BRAF fusion gene, a first probe capable ofspecifically recognizing the 5′-terminal genomic region of the BRAFgene, and a second probe capable of specifically recognizing the3′-terminal genomic region of the BRAF gene, or a first probe capable ofspecifically recognizing the 5′-terminal genomic region of the PXN orGMDS gene, and a second probe capable of specifically recognizing the3′-terminal genomic region of the PXN or GMDS gene (preferably a firstprobe capable of specifically recognizing the 5′-terminal genomic regionof the BRAF gene, and a second probe capable of specifically recognizingthe 3′-terminal genomic region of the BRAF gene) can be used incombination as the probes capable of using in the FISH split assay.

[Detection of mRNA]

The detection of the mRNA may be performed by analyzing the mRNA itselfby Northern hybridization or the like or may be performed by analyzingcomplementary DNA (cDNA) synthesized with the mRNA as a template by amethod well known to those skilled in the art.

A sequencing technique can be suitably used in the detection of the RNA.The sequencing preferably employs a next-generation sequencer (see e.g.,Metzker M L, Nat Rev Genet. 2010 January; 11 (1): 31-46) inconsideration of analysis efficiency. Examples of the next-generationsequencer can include MiSeq/HiSeq from Illumina, Inc., SOLiD System fromLife Technologies Corp., and 454 Sequencing System (GS FLX+/GS Junior)from F. Hoffmann-La Roche, Ltd. In the sequencing, the efficiency ofanalysis can be improved by enriching a region likely to contain thefusion gene using a gene amplification reaction method mentioned later,a sequencing capture technique or the like. Examples of the sequencingcapture technique can include Roche NimbleGen from F. Hoffmann-La Roche,Ltd. and Sure Select from Agilent Technologies, Inc.

<Detection by Gene Amplification Reaction Method>

The mRNA can be detected by a gene amplification reaction method using aprimer designed to be capable of specifically amplifying at least apartial polynucleotide of the BRAF fusion gene or the PXN or GMDS fusiongene to be detected. Hereinafter, typical methods for detecting the mRNAwill be illustrated, but are not limited to those described below.

==PCR==

For example, in PCR, the PCR product is analyzed by agarose gelelectrophoresis, and whether or not an amplified fragment having thesize of interest is obtained can be confirmed by ethidium bromidestaining or the like. The obtainment of the amplified fragment havingthe size of interest means the presence of the BRAF fusion gene or thePXN or GMDS fusion gene in the sample obtained from a subject. In thisway, the BRAF fusion gene or the PXN or GMDS fusion gene can bedetected.

The method for detecting the BRAF fusion gene or the PXN or GMDS fusiongene according to the present invention comprises the step of amplifyinga particular polynucleotide in a sample obtained from a subject throughgene amplification reaction and preferably further comprises the step ofdetecting whether or not an amplified fragment having the size ofinterest is obtained.

PCR is suitable for quantitatively detecting the BRAF fusion gene or thePXN or GMDS fusion gene.

Thus, as described in the preceding section <Aspect (1-b) of detectingBRAF fusion gene>, the BRAF fusion gene can be detected by specificallydetecting the respective expression levels of the 5′-terminal region andthe 3′-terminal region of the BRAF gene and determining the ratiobetween the expression levels. Alternatively, the BRAF fusion gene canbe detected by specifically detecting the respective expression levelsof the 5′-terminal region and the 3′-terminal region of the partnergene, different from the BRAF gene, constituting the BRAF fusion genetogether with the BRAF gene, and determining the ratio between theexpression levels.

Also, as described in the preceding section <Aspect (1-b) of detectingPXN or GMDS fusion gene>, PCR can be suitably used in a method fordetecting the PXN or GMDS fusion gene, comprising specifically detectingthe respective expression levels of the 5′-terminal region and the3′-terminal region of the PXN or GMDS gene and determining the ratiobetween the expression levels. Alternatively, the PXN or GMDS fusiongene can be detected by specifically detecting the respective expressionlevels of the 5′-terminal region and the 3′-terminal region of thepartner gene, different from the PXN or GMDS gene, constituting the PXNor GMDS fusion gene together with the PXN or GMDS gene, and determiningthe ratio between the expression levels.

PCR and a primer design method for use therein can be performed by thoseskilled in the art according to a method known in the art.

For example, sense and antisense primers designed to be capable ofspecifically amplifying the 5′-terminal region of the BRAF gene, andsense and antisense primers designed to be capable of specificallyamplifying the 3′-terminal region of the BRAF gene can be used.

For example, sense and antisense primers designed to be capable ofspecifically amplifying the 5′-terminal region of the PXN or GMDS gene,and sense and antisense primers designed to be capable of specificallyamplifying the 3′-terminal region of the PXN or GMDS gene can be used.

==Real-Time PCR==

In the detection of the BRAF fusion gene or the PXN or GMDS fusion gene,the more quantitative analysis of PCR can be achieved by further usingPCR amplification monitoring (real-time PCR) (Genome Res., 6 (10), 986,1996) in the process of gene amplification. For example, ABI PRISM 7900(PE Biosystems Japan Ltd.) can be used as the PCR amplificationmonitoring. The real-time PCR is a method known in the art, and anapparatus and a kit therefor are commercially available. The real-timePCR is conveniently performed using these commercially availableproducts.

More specifically, when the BRAF fusion gene is, for example, a PXN- orGMDS-BRAF fusion gene and is detected by using its mRNA as an index, thesense primer (5′-primer or forward primer) is designed from an arbitrarysite derived from the PXN or GMDS gene, and the antisense primer(3′-primer or reverse primer) is designed from an arbitrary site derivedfrom the BRAF gene.

When the PXN or GMDS fusion gene is, for example, a PXN- or GMDS-BRAFfusion gene and is detected by using its mRNA as an index, the senseprimer (5′-primer or forward primer) is designed from an arbitrary sitederived from the PXN or GMDS gene, and the antisense primer (3′-primeror reverse primer) is designed from an arbitrary site derived from theBRAF gene.

==Multiplex PCR==

Multiplex PCR which detects all fusion polynucleotides using onereaction solution by mixing sense primers, as described above,corresponding to each partner gene that constitutes the BRAF fusion geneby its fusion with the BRAF gene and a plurality of fusion points canalso be designed as PCR for detecting the BRAF fusion gene.

Multiplex PCR which detects all fusion polynucleotides using onereaction solution by mixing sense primers, as described above,corresponding to each partner gene that constitutes the PXN or GMDSfusion gene by its fusion with the PXN or GMDS gene and a plurality offusion points can also be designed as PCR for detecting the PXN or GMDSfusion gene.

==Detection by Mass Spectrometry==

In the detection method using the gene amplification reaction methoddescribed above, mass spectrometry described in Japanese PatentLaid-Open No. 2012-100628 can be used for the analysis of the amplifiedfragment.

==Primer Set for Use in Detection==

The primer set for use in the detection method of the present inventionfor detecting the BRAF fusion gene is not particularly limited as longas the primer set can specifically amplify at least a portion of theBRAF fusion gene to be detected and permits detection of the BRAF fusiongene. Those skilled in the art can design the primer set on the basis ofthe nucleotide sequence of the polynucleotide to be detected.

The primer set for use in the detection method of the present inventionfor detecting the PXN or GMDS fusion gene is not particularly limited aslong as the primer set can specifically amplify at least a portion ofthe PXN or GMDS fusion gene to be detected and permits detection of thePXN or GMDS fusion gene. Those skilled in the art can design the primerset on the basis of the nucleotide sequence of the polynucleotide to bedetected.

The primers for the PCR amplification monitoring can be designed usingprimer design software (e.g., Primer Express; PE Biosystems Japan Ltd.)or the like. Since increase in the size of a PCR product deterioratesamplification efficiency, the sense primer and the antisense primer areappropriately set such that the size of an amplification product is 1 kbor smaller when mRNA or cDNA is to be amplified.

<Detection by Hybridization>

The mRNA can be detected by hybridization using a probe which hybridizesat least a partial polynucleotide of the BRAF fusion gene or the PXN orGMDS fusion gene to be detected.

Examples of the detection using the hybridization technique includeNorthern hybridization, dot blot, DNA microarray, and RNA protection.

==Probe (for mRNA)==

The probe for use in hybridization is preferably a probe whichhybridizes under stringent conditions (preferably under more stringentconditions) to at least a portion of the BRAF fusion gene or the PXN orGMDS fusion gene or a complementary strand thereof.

<Detection of Fusion Protein>

Any technique well known to those skilled in the art for use in proteinanalysis, or any applied technique based on these techniques may be usedas a technique of detecting the BRAF fusion protein or the PXN or GMDSprotein in a sample obtained from a subject.

Examples of the method for use in the detection of the BRAF fusionprotein can include immunoassay, enzyme-linked immunosorbent assay(ELISA), two-antibody sandwich ELISA, fluorescent immunoassay,radioimmunoassay, Western blotting, immunohistochemistry,immunoprecipitation, iAEP (intercalated antibody-enhanced polymer), andFRET using an antibody specifically recognizing the BRAF protein or thepartner protein, different from the BRAF protein, constituting the BRAFfusion protein together with the BRAF protein, or an antibodyspecifically recognizing the BRAF fusion protein. Alternatively, massspectrometry or amino acid sequencing can be used alone or incombination with any of these methods.

Examples of the method for use in the detection of the PXN or GMDSfusion protein can include immunoassay, enzyme-linked immunosorbentassay (ELISA), two-antibody sandwich ELISA, fluorescent immunoassay,radioimmunoassay, Western blotting, immunohistochemistry,immunoprecipitation, iAEP (intercalated antibody-enhanced polymer), andFRET using an antibody specifically recognizing the PXN or GMDS proteinor the partner protein, different from the PXN or GMDS protein,constituting the PXN or GMDS fusion protein together with the PXN orGMDS protein, or an antibody specifically recognizing the PXN or GMDSfusion protein. Alternatively, mass spectrometry or amino acidsequencing can be used alone or in combination with any of thesemethods.

Hereinafter, typical methods for detecting the protein will beillustrated, but are not limited to those described below.

[Typical Approach for Use in Detection]

The detection method using an antibody can conform to a method known inthe art as described above. For example, the following methods can beused.

<Immunohistochemistry>

When the BRAF fusion protein or the PXN or GMDS fusion protein to bedetected is, for example, a PXN- or GMDS-BRAF fusion protein, a tissuesection likely to contain the fusion protein to be detected isimmunostained with an anti-BRAF antibody binding to a polypeptide havingthe C-terminal region of the BRAF protein and an anti-PXN or anti-GMDSantibody binding to a polypeptide having the N-terminal region of thePXN or GMDS protein. The presence of the fusion protein to be detectedcan also be detected by using these antibodies located in proximity asan index. Alternatively, the tissue section is immunostained with anantibody specifically binding to a polypeptide having the N-terminalregion of the BRAF protein and an antibody specifically binding to apolypeptide having the C-terminal region of the BRAF protein. Thepresence of the fusion protein to be detected can also be detected byusing these antibodies localized distant from each other as an index.Alternatively, the tissue section is immunostained with an antibodyspecifically binding to a polypeptide having the N-terminal region ofthe PXN or GMDS protein and an antibody specifically binding to apolypeptide having the C-terminal region of the PXN or GMDS protein. Thepresence of the fusion protein to be detected can also be detected byusing these antibodies localized distant from each other as an index.

Alternatively, the presence of the fusion protein to be detected may bedetected by immunostaining using an antibody specifically binding to apolypeptide containing the fusion point.

<Western Blotting>

When the BRAF fusion protein or the PXN or GMDS fusion protein to bedetected is, for example, a PXN- or GMDS-BRAF fusion protein, a cellextract likely to contain the fusion protein to be detected iselectrophoresed by a method well known to those skilled in the art, andproteins thus isolated from the cell extract are blotted onto amembrane.

Then, the protein-blotted membrane is immunostained with an anti-BRAFantibody binding to a polypeptide having the N-terminal region of theBRAF protein and an anti-PXN or anti-GMDS antibody binding to theC-terminal region of the PXN or GMDS protein. The presence of the fusionprotein to be detected can also be detected by using, as an index, thebinding of the anti-BRAF antibody and the anti-PXN or anti-GMDS antibodyto the desired positions on the membrane.

Alternatively, the presence of the fusion protein to be detected canalso be detected by using an antibody specifically binding to apolypeptide containing the fusion point, and using, as an index, thebinding of the antibody to the desired position on the membrane.

Alternatively, the presence of the fusion protein to be detected canalso be detected by using an anti-BRAF antibody and using, as an index,the binding of the antibody to the PXN- or GMDS-BRAF fusion protein onthe membrane. In this respect, the presence of the fusion protein to bedetected may be detected by using, as an index, the binding of theanti-BRAF antibody to a position different from the predicted positionof the wild-type BRAF protein on the membrane.

The PXN- or GMDS-BRAF fusion protein may be detected using the anti-PXNor anti-GMDS antibody on the same principle as in use of the anti-BRAFantibody.

<Immunoprecipitation>

When the BRAF fusion protein or the PXN or GMDS fusion protein to bedetected is, for example, a PXN- or GMDS-BRAF fusion protein, a cellextract likely to contain the fusion protein to be detected is subjectedto immunoprecipitation using any one of an anti-BRAF antibody binding toa polypeptide having the C-terminal region of the BRAF protein and ananti-PXN or anti-GMDS antibody binding to a polypeptide having theN-terminal region of the PXN or GMDS protein. The presence of the fusionprotein to be detected can also be detected by detection using the otherantibody in the precipitate. As described above, it is preferred tofurther use the detection antibody after the immunoprecipitation and thedetection to confirm that the detected polypeptide has the size of thepolypeptide of interest to be detected.

Alternatively, a cell extract likely to contain the BRAF fusion proteinto be detected is subjected to immunoprecipitation using an anti-BRAFantibody binding to a polypeptide having the C-terminal region of theBRAF protein. The presence of the fusion protein to be detected can alsobe detected by further subjecting the precipitate to mass spectrometryand thereby confirming the presence of a protein that binds to theanti-BRAF antibody and differs in mass from wild-type BRAF.

A cell extract likely to contain the PXN or GMDS fusion protein to bedetected is subjected to immunoprecipitation using an anti-PXN oranti-GMDS antibody binding to a polypeptide having the N-terminal regionof the PXN or GMDS protein. The presence of the fusion protein to bedetected can also be detected by further subjecting the precipitate tomass spectrometry and thereby confirming the presence of a protein thatbinds to the anti-PXN or anti-GMDS antibody and differs in mass fromwild-type PXN or GMDS.

[Antibody for Use in Detection]

The antibody for use in the detection method according to the presentinvention is not particularly limited as long as the antibodyspecifically binds to the desired site in the BRAF fusion protein or thePXN or GMDS fusion protein. The antibody may be a monoclonal antibody ormay be a polyclonal antibody. A monoclonal antibody and a polyclonalantibody may be used in combination. The antibody may be animmunoglobulin itself or may be an antibody fragment, for example, Fab,Fab′, F(ab′)₂, or Fv, which retains the ability to bind to an antigen.Any labeling or signal amplification method well known to those skilledin the art may be used for the detection of antibody binding.

<Labeling Approach>

In the gene (genomic DNA, mRNA, cDNA, etc.) and protein detectionmethods described above, probes, primers, amplification products,antibodies, etc. can be labeled by use of a technique known in the art.Examples thereof can include fluorescent labeling, chemiluminescentlabeling, radiolabeling, enzymatic labeling, biotinylation, and avidinlabeling.

In the detection method using a probe, the labeling method for labelingthe probe can conform to a method known in the art as described above.For example, in the case of preparing a labeled nucleic acid probe froma BAC clone, an approach known in the art such as nick translation orrandom priming can be used. In this respect, the probe can bebiotinylated with biotin-dUTP (e.g., manufactured by Roche AppliedScience) and further labeled by treatment with a phosphor, aradioisotope, an enzyme, or the like bound with avidin.

In the detection method using an antibody, the labeling method forlabeling the antibody can conform to a method known in the art asdescribed above. Examples thereof include the following labelingmethods.

[iAEP (Intercalated Antibody-Enhanced Polymer)]

Staining sensitivity can be enhanced by incorporating an intercalatingantibody between a first antibody binding to the protein to be detectedand a polymer reagent (Takeuchi et al., Clin Cancer Res, 2009 May 1; 15(9): 3143-3149).

[Fluorescence Resonance Energy Transfer (FRET)]

For example, a probe based on the FRET phenomenon (FRET probe) can beused as an approach of detecting the proximity of two antibodies. One ofthe antibodies may be labeled with a donor fluorescent material (CFP,etc.), and the other antibody may be labeled with an acceptorfluorescent material (YFP, etc.). In this case, YFP becomes in anexcided state by the FRET phenomenon when these fluorescent materialsare positioned sufficiently close to each other. YEP restores its groundstate by emitting fluorescence. The two antibodies located in proximitycan be detected by detecting this fluorescence.

Determination of Applicable Subject to Treatment with BRAF-InhibitingSubstance

When the BRAF fusion gene to be detected or the BRAF fusion protein tobe detected by the detection method of the present invention is detectedfrom a sample obtained from a subject, the subject is a subject(patient) having a BRAF fusion-positive cancer and is an applicablesubject to treatment with a BRAF-inhibiting substance.

Determination of Applicable Subject to Treatment with PXN- orGMDS-Inhibiting Substance

When the PXN or GMDS fusion gene to be detected or the PXN or GMDSfusion protein to be detected by the detection method of the presentinvention is detected from a sample obtained from a subject, the subjectis a subject (patient) having a PXN or GMDS fusion-positive cancer andis an applicable subject to treatment with a PXN- or GMDS-inhibitingsubstance.

Kit for Detection

The kit for detection of the present invention includes a kit for thedetection of the BRAF fusion gene to be detected, or a kit for thedetection of the BRAF fusion protein to be detected.

The kit for detection of the present invention includes a kit for thedetection of the PXN or GMDS fusion gene to be detected, or a kit forthe detection of the PXN or GMDS fusion protein to be detected.

The kit for the detection of the BRAF fusion gene to be detected or thekit for the detection of the PXN or GMDS fusion gene to be detectedaccording to the present invention comprises probes that can be used inFISH fusion assay or FISH split assay in the detection method of thepresent invention, or sense and antisense primers designed to be capableof specifically amplifying at least a portion of the BRAF fusion gene orthe PXN or GMDS fusion gene to be detected in the detection method ofthe present invention. The set of sense and antisense primers is a setof polynucleotides that function as primers for the amplification of apolynucleotide which is at least a partial polynucleotide of the BRAFfusion gene or the PXN or GMDS fusion gene and is to be amplified.

The kit for the detection of the BRAF fusion protein or the PXN or GMDSfusion protein to be detected according to the present inventioncomprises an antibody that can be used in the detection method of thepresent invention.

<Probe>

The kit for the detection of the BRAF fusion gene of the presentinvention can comprise one type or two or more types in combination ofprobe(s) which hybridizes under stringent conditions to at least apartial polynucleotide of the BRAF fusion gene or a complementary strandthereof and permits detection of the BRAF fusion gene.

The kit for the detection of the PXN or GMDS fusion gene of the presentinvention can comprise one type or two or more types in combination ofprobe(s) which hybridizes under stringent conditions to at least apartial polynucleotide of the PXN or GMDS fusion gene or a complementarystrand thereof and permits detection of the PXN or GMDS fusion gene.

Examples of the probe can include any one or more types of probesdescribed in the preceding section

Technique for Use in Detection Method

When the BRAF fusion gene or the PXN or GMDS fusion gene is, forexample, a PXN- or GMDS-BRAF fusion gene, the kit may comprise only oneor more types (preferably two or more types) of probes which hybridizeto the polynucleotide derived from the BRAF gene, or only one or moretypes (preferably two or more types) of probes which hybridize to thepolynucleotide derived from the PXN or GMDS gene, may comprise both ofone or more types of probes which hybridize to the polynucleotidederived from the BRAF gene, and one or more types of probes whichhybridize to the polynucleotide derived from the PXN or GMDS gene, ormay comprise one or more types of probes which hybridize to apolynucleotide containing the fusion point of the BRAF fusion gene, orone or more types of probes which hybridize to a polynucleotidecontaining the fusion point of the PXN or GMDS fusion gene.

<Primer Set>

The kit for the detection of the BRAF fusion gene of the presentinvention can comprise one primer set or two or more primer sets incombination that can specifically amplify at least a portion of the BRAFfusion gene and permits detection of the BRAF fusion gene.

The kit for the detection of the PXN or GMDS fusion gene of the presentinvention can comprise one primer set or two or more primer sets incombination that can specifically amplify at least a portion of the PXNor GMDS fusion gene and permits detection of the PXN or GMDS fusiongene.

Examples of the primer set can include any one or more types of primersets described in the preceding section <<Aspect of detection method ofpresent invention>> or <<Technique for use in detection method>>.

The primer set of the present invention preferably includes

(1) a primer set for detecting a fusion gene of BRAF gene with PXN orGMDS gene, comprising an antisense primer designed from a polynucleotidemoiety encoding the BRAF protein and a sense primer designed from apolynucleotide moiety encoding the PXN or GMDS protein, wherein theantisense primer consists of a nucleic acid molecule (preferably anucleic acid molecule of at least 16 bases) which anneals understringent conditions (preferably under more stringent conditions) to the“polynucleotide to be detected”, and the sense primer consists of anucleic acid molecule (preferably a nucleic acid molecule of at least 16bases) which anneals under stringent conditions (preferably under morestringent conditions) to a complementary strand of the “polynucleotideto be detected”.

In a more specific aspect of the primer set (1), the primer set of thepresent invention includes the following primer sets (2) to (5):

(2) a primer set of a sense primer consisting of an oligonucleotide ofat least 16 consecutive nucleotides arbitrarily selected from nucleotidepositions 1 to 962 of SEQ ID NO: 1 (PXNex6-BRAFex11), and an antisenseprimer consisting of an oligonucleotide complementary to anoligonucleotide of at least 16 consecutive nucleotides arbitrarilyselected from nucleotide positions 963 to 2067 of SEQ ID NO: 1;(3) a primer set of a sense primer consisting of an oligonucleotide ofat least 16 consecutive nucleotides arbitrarily selected from nucleotidepositions 1 to 372 of SEQ ID NO: 3 (GMDSex1-BRAFex9), and an antisenseprimer consisting of an oligonucleotide complementary to anoligonucleotide of at least 16 consecutive nucleotides arbitrarilyselected from nucleotide positions 373 to 1651 of SEQ ID NO: 3;(4) the following primer set for detecting the BRAF fusion gene shown inSEQ ID NO: 1:

PXN-441F: (SEQ ID NO: 8) CCTGCTGCTGGAACTGAAC BRAF-1444R: (SEQ ID NO: 9)CTGCCACATCACCATGCCACTand(5) the following primer set for detecting the BRAF fusion gene shown inSEQ ID NO: 3:

GMDS-1F: (SEQ ID NO: 10) GACATGGCACACGCACCG BRAF-1444R: (SEQ ID NO: 9)CTGCCACATCACCATGCCACT

As described in the preceding section ==PCR== in <Detection by geneamplification reaction method>, the primer set of the present inventionmay be a primer set for detecting the expression levels of the5′-terminal region and the 3′-terminal region of the BRAF gene, or aprimer set for detecting the expression levels of the 5′-terminal regionand the 3′-terminal region of the partner gene that constitutes thefusion gene together with the BRAF gene.

In these primer sets (1) to (5), the interval between the positions atwhich the sense primer and the antisense primer are selected ispreferably 1 kb or smaller, or the size of an amplification productobtained by amplification using the sense primer and the antisenseprimer is preferably 1 kb or smaller.

The primer of the present invention has a chain length of usually 15 to40 bases, preferably 16 to 24 bases, more preferably 18 to 24 bases,particularly preferably 20 to 24 bases.

The primer set of the present invention can be used for amplifying anddetecting the polynucleotide to be detected in the detection method ofthe present invention. Each primer contained in the primer set of thepresent invention can be produced by, for example, a chemical synthesismethod, though the production method is not particularly limitedthereto.

<Antibody>

The kit for the detection of the BRAF fusion protein of the presentinvention can comprise one type of antibody or two or more types ofantibodies in combination specifically binding to an arbitrary site inthe BRAF fusion protein. Specific examples thereof can include theantibodies described in the preceding section

<Detection of Fusion Protein>.

The kit for the detection of the PXN or GMDS fusion protein of thepresent invention can comprise one type of antibody or two or more typesof antibodies in combination specifically binding to an arbitrary sitein the PXN or GMDS fusion protein. Specific examples thereof can includethe antibodies described in the preceding section <Detection of fusionprotein>.

When the BRAF fusion protein or the PXN or GMDS fusion protein is, forexample, a PXN- or GMDS-BRAF fusion protein, the kit may comprise onlyone or more types (preferably two or more types) of antibodies bindingto the polypeptide derived from the BRAF protein, or only one or moretypes (preferably two or more types) of antibodies binding to thepolypeptide derived from the PXN or GMDS protein, may comprise both ofone or more types of antibodies binding to the polypeptide derived fromthe BRAF protein, and one or more types of antibodies binding to thepolypeptide derived from the PXN or GMDS protein, or may comprise one ormore types of antibodies binding to a polypeptide containing the fusionpoint of the BRAF fusion protein, or one or more types of antibodiesbinding to a polynucleotide containing the fusion point of the PXN orGMDS fusion gene.

Inhibiting Substance Screening Method

<Step of Screening for Substance that Inhibits Polypeptide>

The inhibiting substance screening method of the present invention canscreen for a substance that inhibits the polypeptide to be detected andcomprises the steps of:

(1) contacting the polypeptide to be detected or a cell expressing thepolypeptide with a test substance;(2) analyzing whether or not to inhibit the polypeptide; and(3) selecting the substance that inhibits the polypeptide.

In the present specification, the phrase “inhibition of the polypeptide”includes the inhibition of the activity of the polypeptide and theinhibition of the expression of the polypeptide. The “inhibition” meansthe inhibition of at least a portion.

<Inhibiting Substance Screening Step and Index Therefor>

The screening method of the present invention includes

(A) a method using a purified or crude polypeptide and using the invitro activity inhibition of the polypeptide as an index,(B) a method using a cell expressing the polypeptide and using theactivity inhibition of the polypeptide as an index, and(C) a method using a cell expressing the polypeptide and using theexpression inhibition of the polypeptide as an index.

[(A) Method Using Purified or Crude Polypeptide and Using ActivityInhibition as Index]

The method (A) includes a method comprising the steps of: adding a testsubstance to the polypeptide in vitro for contact; analyzing whether ornot the test substance inhibits the activity of the polypeptide bycomparison with a control (polypeptide not contacted with the testsubstance); and selecting a substance that has inhibited the activity ofthe polypeptide.

The polypeptide activity can be measured in vitro by use of a kinaseactivity measurement method known in the art. For example, the amount ofADP formed through kinase reaction may be used as an index, or thetyrosine phosphorylation level of the polypeptide may be used as anindex. A commercially available kinase activity measurement kit can alsobe used.

[(B) Method Using Polypeptide-Expressing Cell and Using ActivityInhibition as Index]

The method (B) includes a method comprising the steps of: adding a testsubstance to a cell expressing the polypeptide for contact; analyzingwhether or not the test substance inhibits the activity of thepolypeptide by comparison with a control (cell not contacted with thetest substance); and selecting a substance that has inhibited theactivity of the polypeptide.

The polypeptide activity can be measured in the cell by use of a kinaseactivity measurement method known in the art. For example, the amount ofADP formed through kinase reaction may be used as an index, or thetyrosine phosphorylation level of the polypeptide may be used as anindex. A commercially available kinase activity measurement kit can alsobe used.

[(C) Method Using Polypeptide-Expressing Cell and Using ExpressionInhibition as Index]

The method (C) includes a method comprising the steps of: adding a testsubstance to a cell expressing the polypeptide for contact; analyzingwhether or not the test substance inhibits the expression of thepolypeptide by comparison with a control (cell not contacted with thetest substance); and selecting a substance that has inhibited theexpression of the polypeptide.

The expression of the polypeptide in the cell can be analyzed bymeasuring a protein or mRNA level. The protein level can be measured byuse of, for example, ELISA or immunoblot. The mRNA level can be measuredby use of, for example, RT-PCR or Northern blot.

In this context, the BRAF fusion gene is a gene having tumorigenicity.Thus, the polypeptide-inhibiting substance selected by the inhibitingsubstance screening method of the present invention is useful as anactive substance or a candidate substance thereof in a pharmaceuticalcomposition for the treatment of a BRAF fusion-positive cancer. Themethod of the present invention can further comprise, if desired, thestep of confirming that the inhibiting substance has therapeuticactivity against a BRAF fusion-positive cancer.

Also, the PXN or GMDS fusion gene is a gene having tumorigenicity. Thus,the polypeptide-inhibiting substance selected by the inhibitingsubstance screening method of the present invention is useful as atherapeutic drug or a candidate substance thereof for a PXN or GMDSfusion-positive cancer. The method of the present invention can furthercomprise, if desired, the step of confirming that the inhibitingsubstance has therapeutic activity against a PXN or GMDS fusion-positivecancer.

The confirmation step can be carried out using an evaluation systemknown in the art. Examples thereof can include in vitro evaluationsystems using cultured cells and evaluation systems using cancer-bearinganimal models obtained by the transplantation of tumor cells. For thecancer-bearing animal models, a cell line may be temporarily establishedby culture from tumor tissues surgically resected from a patient andthen transplanted to a recipient, or the tumor tissues may be directlytransplanted to a recipient. The cancer-bearing animal model obtained bythe latter approach is known as a PDX (patient-derived xenograft) animalmodel and is preferred for the evaluation system because gene expressionprofiles from repeated passages of subcutaneous tumor tissues are moresimilar to those from primary tumor as compared with the cellline-transplanted animal model.

The polypeptide-expressing cell can also be obtained by transfecting thedesired cells with the polynucleotide of the present invention accordingto a routine method (see e.g., Molecular Cloning: A Laboratory Manual4th Edition (2012), Cold Spring Harbor Laboratory Press). Specifically,the polypeptide-expressing cell (transformed cell) can be obtained, forexample, by inserting cDNA which is the BRAF fusion gene or the PXN orGMDS fusion gene of the present invention to a recombinant vector andfurther transfecting cells with this vector.

Pharmaceutical Composition for Treatment of Cancer, ContainingInhibiting Substance

The pharmaceutical composition for the treatment of a BRAFfusion-positive cancer of the present invention comprises a substancethat inhibits the BRAF fusion gene or a transcript thereof. Thepharmaceutical composition contains, for example, an inhibitingsubstance (e.g., a low-molecular compound, a double-stranded nucleicacid (including siRNA), a protein (including an antibody or an antibodyfragment), a peptide, and other compounds) obtained by the inhibitingsubstance screening method of the present invention as an activeingredient and can contain, if desired, a pharmaceutically acceptablecarrier.

The pharmaceutical composition for the treatment of a PXN or GMDSfusion-positive cancer of the present invention comprises a substancethat inhibits the PXN or GMDS fusion gene or a transcript thereof. Thepharmaceutical composition contains, for example, an inhibitingsubstance (e.g., a low-molecular compound, a double-stranded nucleicacid (including siRNA), a protein (including an antibody or an antibodyfragment), a peptide, and other compounds) obtained by the inhibitingsubstance screening method of the present invention as an activeingredient and can contain, if desired, a pharmaceutically acceptablecarrier.

<Substance that Inhibits BRAF Fusion Gene or Transcript, or PXN or GMDSFusion Gene or Transcript>

Examples of the substance that inhibits the BRAF fusion gene or atranscript thereof can include kinase inhibitors, for example,BRAF-inhibiting substances and substances inhibiting the partner genethat constitutes the fusion gene together with the BRAF gene, or atranscript thereof.

Examples of the substance that inhibits the PXN or GMDS fusion gene or atranscript thereof can include kinase inhibitors, for example, PXN- orGMDS-inhibiting substances and substances inhibiting the partner genethat constitutes the fusion gene together with the PXN or GMDS gene, ora transcript thereof.

[Low-Molecular Compound]

Among the inhibiting substances described above, specific examples ofthe low-molecular compound can include trametinib (Japan Tobacco Inc.),sorafenib (Bayer Healthcare Pharmaceuticals Inc.), dabrafenib(GlaxoSmithKline LLC.), vemurafenib (PLX-4032, Hoffmann-La Roche Inc.),regorafenib (Bayer Healthcare Pharmaceuticals Inc.),N-{2,4-difluoro-3-[(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)carbonyl]phenyl}ethanesulfonamide,N-{3-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)carbonyl]-2,4-difluorophenyl}propane-1-sulfonamide,(1E)-5-(1-piperidin-4-yl-3-pyridin-4-yl-1H-pyrazol-4-yl)-2,3-dihydro-1H-inden-1-oneoxime, GDC-0879((E)-2,3-dihydro-5-[1-(2-hydroxyethyl)-3-(4-pyridinyl)-1H-pyrazol-4-yl]-1H-inden-1-oneoxime), RAF-265(1-methyl-5-(2-(5-(trifluoromethyl)-1H-imidazol-2-yl)pyridin-4-yloxy)-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazol-2-amine),AZ628(3-(2-cyanopropan-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-ylamino)phenyl)benzamide),and their pharmaceutically acceptable salts.

[Double-Stranded Nucleic Acid]

The double-stranded nucleic acid consists of a double-stranded nucleicacid (RNA or DNA) moiety and, preferably, 3′-terminal overhangs of thesense and antisense strands and induces RNAi. RNAi is an evolutionarilyconserved phenomenon and occurs via a double-stranded nucleic acid of 21to 23 bases formed by RNase III endonuclease (Genes Dev. 15, 485-490,2001). Each 3′-overhang is an arbitrary nucleic acid of 1 or 2 bases,preferably 2 bases. The number of bases (21 to 23 bases) described aboveis the number of bases in each strand (sense or antisense strand)including the overhang. The sense strand and the antisense strand mayhave the same number of bases or may have different numbers of bases andpreferably have the same number of bases.

For example, U (uridine), A (adenosine), G (guanosine), or C (cytidine)can be used as a ribonucleic acid constituting the 3′-overhang of thedouble-stranded nucleic acid. For example, dT (deoxythymidine), dA(deoxyadenosine), dG (deoxyguanosine), or dC (deoxycytidine) can be usedas a deoxyribonucleic acid constituting the 3′-overhang.

The double-stranded nucleic acid that can be used as an activeingredient in the pharmaceutical composition of the present invention isnot particularly limited as long as the double-stranded nucleic acid hasan inhibitory effect on the BRAF fusion gene or an inhibitory effect onthe PXN or GMDS fusion gene. For example, the double-stranded moiety canbe designed on the basis of the nucleotide sequence of a polynucleotidecontaining the fusion point, for example, a nucleotide sequenceincluding positions 962 and 963 of SEQ ID NO: 1, or a nucleotidesequence including positions 372 and 373 of SEQ ID NO: 3. Alternatively,the double-stranded moiety can be designed on the basis of thenucleotide sequence of a polynucleotide encoding a kinase moiety. Thedouble-stranded nucleic acid of the present invention can be produced bya routine method (e.g., J. Am. Chem. Soc., 120, 11820-11821, 1998; andMethods, 23, 206-217, 2001). Commission manufacturers of double-strandednucleic acids (e.g., RNAi Inc.) are well known to those skilled in theart and can be used in the production of the double-stranded nucleicacid. Also, the double-stranded nucleic acid can be designed using asiRNA sequence design system (Commercial siDirect®; RNAi Inc.).

[Protein and Antibody]

The antibody that can be used as an active ingredient in thepharmaceutical composition of the present invention is not limited aslong as the antibody inhibits a transcript of the BRAF fusion gene or atranscript of the PXN or GMDS fusion gene, preferably a transcript ofthe PXN- or GMDS-BRAF gene. Examples thereof include antibodiesinhibiting the activity of the BRAF fusion protein or the PXN or GMDSfusion protein, preferably kinase activity.

Polypeptide, Polynucleotide, Vector, and Transformed Cell of PresentInvention

<Polypeptide of Present Invention>

The PXN or GMDS fusion protein, preferably the PXN-BRAF fusion proteinor the GMDS-BRAF fusion protein, to be detected according to thedetection method of the present invention is a novel protein in itself.

The PXN-BRAF fusion protein which is the polypeptide of the presentinvention is preferably any of the following polypeptides (a) to (d):

(a) a polypeptide consisting of the amino acid sequence represented bySEQ ID NO: 2,(b) a polypeptide comprising the amino acid sequence represented by SEQID NO: 2, and having tumorigenicity,(c) a polypeptide comprising an amino acid sequence with 80% or higheridentity to the amino acid sequence represented by SEQ ID NO: 2, andhaving tumorigenicity, and(d) a polypeptide comprising an amino acid sequence derived from theamino acid sequence represented by SEQ ID NO: 2 by the deletion,substitution, and/or insertion of one or several amino acids, and havingtumorigenicity.

The GMDS-BRAF fusion protein which is the polypeptide of the presentinvention is preferably any of the following polypeptides (a) to (d):

(a) a polypeptide consisting of the amino acid sequence represented bySEQ ID NO: 4,(b) a polypeptide comprising the amino acid sequence represented by SEQID NO: 4, and having tumorigenicity,(c) a polypeptide comprising an amino acid sequence with 80% or higheridentity to the amino acid sequence represented by SEQ ID NO: 4, andhaving tumorigenicity, and(d) a polypeptide comprising an amino acid sequence derived from theamino acid sequence represented by SEQ ID NO: 4 by the deletion,substitution, and/or insertion of one or several amino acids, and havingtumorigenicity.

<Polynucleotide of Present Invention>

The PXN or GMDS fusion gene, preferably the PXN-BRAF fusion gene or theGMDS-BRAF fusion gene, to be detected according to the detection methodof the present invention is a novel gene in itself.

The polynucleotide of the present invention is not particularly limitedas long as the polynucleotide encodes the polypeptide (i.e., the PXN orGMDS fusion protein, preferably the PXN-BRAF fusion protein or theGMDS-BRAF fusion protein) of the present invention (i.e., the PXN orGMDS fusion gene, preferably the PXN-BRAF fusion gene or the GMDS-BRAFfusion gene). The “polynucleotide encoding the PXN-BRAF fusion protein”may be a polynucleotide consisting only of a coding region in thePXN-BRAF fusion gene, may be the full-length genomic DNA of the PXN-BRAFfusion gene, or may be mRNA or cDNA of the PXN-BRAF fusion gene. Also,the “polynucleotide encoding the GMDS-BRAF fusion protein” may be apolynucleotide consisting only of a coding region in the GMDS-BRAFfusion gene, may be the full-length genomic DNA of the GMDS-BRAF fusiongene, or may be mRNA or cDNA of the GMDS-BRAF fusion gene.

<Vector of Present Invention>

The vector of the present invention is not particularly limited as longas the vector comprises the polynucleotide of the present invention. Thevector of the present invention can be prepared by integrating thepolynucleotide into an appropriate vector that can transform eukaryoticor prokaryotic host cells. The vector can comprise a sequence(s)necessary for the expression of the polynucleotide, for example, apromoter and an enhancer and can further comprise a sequence necessaryfor the confirmation of transfection of a host, for example, a drugresistance gene.

<Transformed Cell of Present Invention>

The transformed cell of the present invention can be prepared bytransforming appropriate host cells, for example, eukaryotic orprokaryotic host cells with the vector of the present invention. Thetransformed cell of the present invention can be used for producing thepolypeptide of the present invention.

EXAMPLES

Hereinafter, the present invention will be specifically described withreference to Examples. However, these Examples are not intended to limitthe scope of the present invention.

In embodiments and Examples, methods described in standard protocolssuch as J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecularcloning, a laboratory manual (3rd edition), Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (2001); and F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.),Current Protocols in Molecular Biology, John Wiley & Sons Ltd., ormethods modified or altered from these methods are used unless otherwisespecified. In the case of using commercially available reagent kits ormeasurement apparatuses, protocols attached thereto are used unlessotherwise specified.

[Example 1] Detection of BRAF Gene Abnormality in Clinical Samples byFISH

A method is known which involves labeling 5′-terminal and 3′-terminalregions of the gene of interest with different dyes and observing thetranslocation or inversion, etc. of the gene. This method, one type ofFISH, is called split assay. In the split assay, the 5′-terminal and3′-terminal regions of the gene of interest to be examined for itschromosomal translocation or inversion, etc. are respectively stainedwith probes labeled with different fluorescent dyes. For example, theseterminal regions are labeled with two types of fluorescent probes, aTexas Red (red)-labeled probe and a FITC (green)-labeled probe. As aresult, one yellow signal (derived from green and red signals present inproximity) is detected under normal conditions (where no fusion gene isformed), while distant green and red signals are detected in thepresence of translocation or inversion, etc.

BRAF gene abnormality in clinical samples was detected by the FISH splitassay. Colorectal cancer tissues that were surgically resected, fixed in20% formalin, and embedded in paraffin were sliced into a thickness of 4μm, and these slices were placed on glass slides to prepare pathologicalsections. FISH was conducted according to the method described in theliterature (Takeuchi K, Choi Y L, Soda M, Inamura K, Togashi Y, HatanoS, Enomoto M, Takada S, Yamashita Y, Satoh Y, Okumura S, Nakagawa K,Ishikawa Y, Mano H. Multiplex reverse transcription-PCR screening forEML4-ALK fusion transcripts. Clin Cancer Res. 2008; 14: 6618-6624). Theprepared unstained sections were treated with Histology FISH AccessoryKit (Dako/Agilent Technologies, Inc.), followed by hybridization using ared (Texas Red) fluorescence-labeled BAC (bacterial artificialchromosome) clone (clone No. RP11-159M20) covering the 5′-terminalregion of the BRAF gene, and a green (FITC) fluorescence-labeled BACclone (clone No. RP11-759K14 or CTD-2337E12) covering the 3′-terminalregion of the BRAF gene. Subsequently, the sections were further stainedwith 4,6-diamino-2-phenylindole. A fluorescence microscope BX51 (OlympusCorp.) was used in fluorescent observation. Sections suggesting genomestructure abnormality were found by observed distant green and redsignals. The study on approximately 1500 pathological samples identifiedtwo cases (colon cancer patient) suggesting genome structure abnormalityin the BRAF gene region.

[Example 2] Gene Identification of BRAF Fusion Polynucleotide inClinical Samples

RNA derived from each tissue that suggested BRAF genome structureabnormality by the FISH analysis was used as a template in capturesequencing using Sure Select Human Kinome RNA Kit (Agilent Technologies,Inc.) and Miseq (Illumina, Inc.) to examine a gene located upstream ofthe kinase region of BRAF gene. The results demonstrated that a portionof PXN gene or GMDS gene is fused upstream of the BRAF gene kinaseregion.

[Example 3] Gene Isolation from PXN-BRAF and GMDS-BRAF FusionPolynucleotides in Clinical Samples

cDNA derived from each colorectal cancer clinical sample in which BRAFgenome structure abnormality was suggested and a partner of the fusiongene was identified by the FISH analysis was used as a template in PCRusing DNA polymerase (PrimeStar HS DNA polymerase), and eachamplification product was cloned into pT7Blue-2. A forward primerGMDS-5′UTR (SEQ ID NO: 5) and a reverse primer BRAF-3′UTR (SEQ ID NO: 6)were used in combination as a primer set for isolation of the PXN-BRAFfusion polynucleotide gene. Also, a forward primer GMDS-5′UTR (SEQ IDNO: 7) and a reverse primer BRAF-3′UTR (SEQ ID NO: 6) were used incombination as a primer set for isolation of the GMDS-BRAF fusionpolynucleotide gene.

As a result of sequencing the obtained amplification products (1914 bpand 1440 bp), a polynucleotide consisting of nucleotide sequences fromstart codon ATG to exon 6 of the PXN gene and from exon 11 to stop codonat exon 18 of the BRAF gene (PXNex6-BRAFex11; SEQ ID NO: 1) was able tobe obtained. Also, a polynucleotide consisting of nucleotide sequencesfrom start codon ATG to exon 1 of the GMDS gene and from exon 9 to stopcodon at exon 18 of the BRAF gene (GMDSex1-BRAFex9; SEQ ID NO: 3) wasable to be obtained.

The amino acid sequence (SEQ ID NO: 2) encoded by PXNex6-BRAFex11 wasconfirmed to have an amino acid substitution at one position (S73G) bycomparison with the registered amino acid sequence of PXN (Ensembledatabase, Protein ID: ENSP00000288602). For the amino acid substitution,the first amino acid (e.g., “S” in S73G) means the amino acid in theregistered amino acid sequence; the amino acid position subsequentthereto (e.g., “73” in S73G) means the amino acid position in SEQ ID NO:2; and the final amino acid (e.g., “G” in S73G) means the amino acid inthe amino acid sequence represented by SEQ ID NO: 2. C at position 348,which results in the amino acid substitution, in the nucleotide sequencerepresented by SEQ ID NO: 1 has been reported as a single-nucleotidepolymorphism (rs4767884).

TABLE 1 #1 Primer name #2 Sequence #3 SEQ ID NO:

[Example 4] Detection of PXN-BRAF Fusion Gene and GMDS-BRAF Fusion Gene

cDNA from each fusion gene was detected by RT-PCR, which directlyamplified a fusion part-containing region using the primers shown inTable 1, to show the presence of cDNA of the fusion gene in cancertissues. Specifically, for the PXN-BRAF fusion gene, the sample-derivedRNA template was subjected to PCR using a forward primer PXN-441F (SEQID NO: 8) designed on the PXN gene and a reverse primer BRAF-1444R (SEQID NO: 9) designed on the BRAF gene. As a result of electrophoresing theamplification product, a band having a size (521 bp) predicted from theprimer set positions was observed. For the GMDS-BRAF fusion gene, thesample-derived RNA template was subjected to PCR using a forward primerGMDS-1F (SEQ ID NO: 10) designed on the GMDS gene and a reverse primerBRAF-1444R (SEQ ID NO: 9) designed on the BRAF gene. As a result ofelectrophoresing the amplification product, a band having a size (396bp) predicted from the primer set positions was observed. The fusiongenes were found detectable using clinical samples by designing primerson their genes.

[Example 5] Detection of PXN-BRAF Fusion Gene and GMDS-BRAF Fusion Genein Clinical Samples by FISH Fusion Assay

In order to confirm the partners in the fusion genes were fused on thegenome, detection was performed by FISH fusion assay.

In the FISH fusion assay, two gene regions of interest that come inproximity due to chromosomal translocation or inversion, etc. arerespectively stained with probes labeled with different fluorescentdyes. For example, these gene regions are labeled with two types offluorescent probes, a Texas Red (red)-labeled probe and a FITC(green)-labeled probe. As a result, their respective red and greensignals (distant red and green signals) are detected under normalconditions (where no fusion gene is formed), while a yellow signalresulting from the overlap of the red and green signals is detected whenthe two gene regions are located in proximity in the presence oftranslocation or inversion, etc.

Specifically, for the PXN-BRAF fusion gene, a red (Texas Red)fluorescence-labeled BAC clone (clone No. CTD-2308L15 or CTD-3139J18)covering the 5′-terminal region of the PXN gene, and a green (FITC)fluorescence-labeled BAC clone (clone No. RP11-759K14 or CTD-2337E12)covering the 3′-terminal region of the BRAF gene were used incombination.

For the GMDS-BRAF fusion gene, a red (Texas Red) fluorescence-labeledBAC clone (clone No. RP1-33B19, RP1-80B9, or RP1-136B1) covering the5′-terminal region of the GMDS gene, and a green (FITC)fluorescence-labeled BAC clone (clone No. RP11-759K14 or CTD-2337E12)covering the 3′-terminal region of the BRAF gene were used incombination.

A fluorescence microscope BX51 (Olympus Corp.) was used in fluorescentobservation. As a result of conducting the fusion assay using thepathological sections found positive to the fusion genes in Example 4, asignal (yellow) from the 5′-terminal region of the PXN gene and the3′-terminal region of the BRAF gene located in proximity, or a signal(yellow) from the 5′-terminal region of the GMDS gene and the3′-terminal region of the BRAF gene located in proximity was observed,confirming that the fusion genes were formed by chromosomaltranslocation or inversion, etc.

This method was found usable as a method for detecting the presence ofthese fusion genes.

[Example 6] Study on Tumorigenicity of PXN-BRAF and GMDS-BRAF FusionPolypeptides

In this Example, the PXN-BRAF fusion gene of SEQ ID NO: 1 obtained froma colorectal cancer clinical sample in Example 3 was used as cDNAencoding the PXN-BRAF fusion polypeptide. Also, the GMDS-BRAF fusiongene of SEQ ID NO: 3 obtained from a colorectal cancer clinical samplein Example 3 was used as cDNA encoding the GMDS-BRAF fusion polypeptide.The tumorigenicity of each fusion polypeptide was studied. Each cDNAdescribed above was inserted to an expression vector pLenti6(Invitrogen® (Life Technologies Corp.)), and mouse fibroblast lineNIH3T3 cells were transfected with the resulting pLenti6-PXN-BRAF orpLenti6-GMDS-BRAF and cultured for 7 days. As a result, transformed fociwere observed, as shown in FIGS. 1 (PXN-BRAF) and 2 (GMDS-BRAF). Such atransformed focus was observed in neither NIH3T3 cells treated only witha transfection reagent (control) nor NIH3T3 cells transfected with LacZ(not shown). In short, the transformed focus was observed only when thecells were transfected with the PXN-BRAF or GMDS-BRAF fusion polypeptideexpression vector.

The NIH3T3 cells transfected with the PXN-BRAF or GMDS-BRAF fusionpolypeptide expression vector, the NIH3T3 cells treated only with atransfection reagent (control), and the NIH3T3 cells transfected withLacZ were subcutaneously inoculated at 1×10⁶ cells each/body to nudemice. As a result, tumor formation was confirmed in the mouse inoculatedwith the NIH3T3 cells transfected with the fusion polypeptide expressionvector. No tumor formation was confirmed in the mouse inoculated withthe NIH3T3 cells treated only with a transfection reagent (control). Thetumor sizes in both the mice on inoculation day 1 and later are shown inFIG. 3.

From these results, the PXN-BRAF or GMDS-BRAF fusion polypeptide hadtumorigenicity, showing that the PXN-BRAF or GMDS-BRAF fusionpolynucleotide is a causative gene of cancers.

As a result of transfecting NIH3T3 cells with cDNA encoding thefull-length BRAF polypeptide, no transformed focus was observed,confirming that the full-length BRAF polypeptide is free fromtumorigenicity.

[Example 7] Study on Sensitivity of PXN-BRAF or GMDS-BRAF FusionPolypeptide-Expressing Cell to BRAF Inhibitor

A mouse lymphoid cell line Ba/F3 is a cell line dependent on a growthfactor IL-3 and requires IL-3 for its growth. The Ba/F3 cells are knownto become able to grow without the addition of IL-3, when transfectedwith an oncogene (e.g., a tyrosine kinase fusion gene) (Daley G Q andBaltimore D. Proc Natl Acad Sci USA. 1988 December; 85 (23): 9312-9316).

In this Example, the sensitivity of parent line Ba/F3 cells and Ba/F3cells transfected with pLenti6-PXN-BRAF or pLenti6-GMDS-BRAF prepared inExample 6 to a BRAF inhibitor (trametinib) was studied by adding apredetermined concentration of the BRAF inhibitor to 2000 cells andcounting the number of cells after culture for 72 hours. For a morespecific testing method, see, for example, the literature of Katayama etal. (Katayama R et al., Sci Transl Med, 2012 (4): 120ra17).

The results are shown in FIG. 4. The BRAF inhibitor had no influence onthe ability to grow of the parent line Ba/F3 cells (IL-3concentration=0.5 ng/mL) as a control unless the concentration of theBRAF inhibitor exceeded an excessive level that exerted cytotoxicity. Onthe other hand, the cell growth of the Ba/F3 cells transfected withpLenti6-PXN-BRAF or pLenti6-GMDS-BRAF (without the addition of IL-3) wassignificantly inhibited by the BRAF inhibitor in aconcentration-dependent manner as to both the fusion genes.

These results show that the BRAF inhibitor is effective for thetreatment of PXN-BRAF or GMDS-BRAF fusion gene-positive cancer patients,and also shows that this evaluation system using the Ba/F3 cellstransfected with pLenti6-PXN-BRAF or pLenti6-GMDS-BRAF can be used forscreening for a drug effective for the treatment of such fusiongene-positive cancer patients.

[Example 8] Study on Phosphorylation Inhibition of PXN-BRAF or GMDS-BRAFFusion Polypeptide by BRAF Inhibitor in PXN-BRAF or GMDS-BRAF FusionPolypeptide-Expressing Cell

In order to confirm that the growth inhibition of the PXN-BRAF orGMDS-BRAF fusion polypeptide-expressing cells by the BRAF inhibitorconfirmed in Example 7 was attributed to the inhibition of the kinaseactivity of the PXN-BRAF or GMDS-BRAF fusion polypeptide against MEK(MAPK/ERK kinase), each cultured cell-derived extract treated with theBRAF inhibitor was subjected to Western blotting.

The results are shown in FIG. 5. An anti-phosphorylated MEK antibody wasused in the detection of phosphorylated MEK (pMEK in FIG. 5), and ananti-MEK antibody was used in the detection of MEK. The polypeptidelevel of MEK rarely differed between the presence and absence of thetreatment with the BRAF inhibitor (and among the treatmentconcentrations). On the other hand, the MEK phosphorylation of thePXN-BRAF or GMDS-BRAF fusion polypeptide was significantly decreased bythe treatment with the BRAF inhibitor in a concentration-dependentmanner, confirming that the MEK phosphorylation of the PXN-BRAF orGMDS-BRAF fusion polypeptide is inhibited by the inhibition of thekinase activity of the PXN-BRAF or GMDS-BRAF fusion polypeptide.

Thus, the present invention demonstrated that a fusion gene of the PXNor GMDS gene with the BRAF gene is present in some digestive organcancer patients, and the gene is responsible for their cancers. Theseresults demonstrated that cancer patients to be treated with a BRAFinhibitor can be selected by detecting the PXN-BRAF or GMDS-BRAF fusiongene, preferably by detecting PXNex6-BRAFex11 or GMDSex1-BRAFex9.

INDUSTRIAL APPLICABILITY

The detection method of the present invention is useful in thedetermination of a BRAF fusion-positive cancer patient. The kit and theprimer set for detection of the present invention can be used in thedetection method. The inhibiting substance screening method of thepresent invention can be used for screening for a substance effectivefor the treatment of the fusion-positive cancer patient. The substanceobtained by the screening can be used as an active ingredient in apharmaceutical composition for the treatment of the fusion-positivecancer. The cancer in a patient determined as the fusion-positive cancerpatient by the detection method can be treated by the administration ofthe substance.

The detection method of the present invention is useful in thedetermination of a PXN or GMDS fusion-positive cancer patient. The kitand the primer set for detection of the present invention can be used inthe detection method. The inhibiting substance screening method of thepresent invention can be used for screening for a substance effectivefor the treatment of the fusion-positive cancer patient. The substanceobtained by the screening can be used as an active ingredient in apharmaceutical composition for the treatment of the fusion-positivecancer. The cancer in a patient determined as the fusion-positive cancerpatient by the detection method can be treated by the administration ofthe substance.

The present invention is described above with reference to particularaspects. However, changes or modifications obvious to those skilled inthe art are included in the scope of the present invention.

Free Text of Sequence Listing

The nucleotide sequences represented by SEQ ID NOs: 5 to 10 of theSequence Listing are synthetic primer sequences.

1. A kit for the detection of a GMDS-BRAF fusion gene, comprising senseand antisense primers designed to be capable of specifically amplifyinga polynucleotide encoding a polypeptide which is a fusion protein of theGMDS protein with the BRAF protein.
 2. The kit for the detection of aGMDS-BRAF fusion gene according to claim 1, comprising sense andantisense primers designed to be capable of specifically amplifying apolynucleotide encoding a polypeptide selected from the group consistingof the following polypeptides (a) to (d): (a) a polypeptide consistingof the amino acid sequence represented by SEQ ID NO: 4; (b) apolypeptide comprising the amino acid sequence represented by SEQ ID NO:4, and having tumorigenicity; (c) a polypeptide comprising an amino acidsequence with 80% or higher identity to the amino acid sequencerepresented by SEQ ID NO: 4, and having tumorigenicity; and (d) apolypeptide comprising an amino acid sequence derived from the aminoacid sequence represented by SEQ ID NO: 4 by the deletion, substitution,and/or insertion of one or several amino acids, and havingtumorigenicity.
 3. A primer set used for the kit according claim 1,comprising an antisense primer consisting of a nucleic acid moleculewhich anneals under stringent conditions to a polynucleotide consistingof the nucleotide sequence represented by SEQ ID NO: 3, and a senseprimer consisting of a nucleic acid molecule which anneals understringent conditions to a complementary strand of the polynucleotide. 4.The primer set according claim 3, comprising a sense primer and anantisense primer selected from the group consisting of the followingsense and antisense primers: a sense primer consisting of anoligonucleotide of at least 16 consecutive nucleotides arbitrarilyselected from nucleotide positions 1 to 372 of SEQ ID NO: 3, and anantisense primer consisting of an oligonucleotide complementary to anoligonucleotide of at least 16 consecutive nucleotides arbitrarilyselected from nucleotide positions 373 to 1651 of SEQ ID NO:
 3. 5. Aprimer set for detecting a fusion gene of BRAF gene with GMDS gene,comprising an antisense primer designed from a polynucleotide moietyencoding the BRAF protein, and a sense primer designed from apolynucleotide moiety encoding the GMDS protein, wherein the antisenseprimer consists of a nucleic acid molecule which anneals under stringentconditions to a polynucleotide according to claim 2, and the senseprimer consists of a nucleic acid molecule which anneals under stringentconditions to a complementary strand of the polynucleotide according toclaim
 2. 6. A method for detecting a GMDS-BRAF fusion gene encoding thefusion protein in a sample obtained from a subject, using the kitaccording to claim
 1. 7. A method for detecting a GMDS-BRAF fusion geneencoding the fusion protein in a sample obtained from a subject, usingthe primer set according to claim
 3. 8. A method for detecting aGMDS-BRAF fusion gene encoding the fusion protein in a sample obtainedfrom a subject, using the primer set according to claim
 5. 9. A kit forthe detection of a GMDS-BRAF fusion gene, comprising probes designed tobe capable of specifically recognizing the presence of GMDS-BRAF fusiongene.
 10. The kit for the detection of a GMDS-BRAF fusion gene accordingto claim 9, comprising a first probe capable of specifically recognizinga 5′-terminal genomic region of a GMDS gene, and a second probe capableof specifically recognizing a 3′-terminal genomic region of the BRAFgene.
 11. The kit for the detection of a GMDS-BRAF fusion gene accordingto claim 9, comprising a first probe capable of specifically recognizinga 5′-terminal genomic region of the GMDS gene, and a second probecapable of specifically recognizing a 3′-terminal genomic region of theGMDS gene.
 12. The kit for the detection of a GMDS-BRAF fusion geneaccording to claim 9, comprising a first probe capable of specificallyrecognizing a 5′-terminal genomic region of the BRAF gene, and a secondprobe capable of specifically recognizing a 3′-terminal genomic regionof the BRAF gene.
 13. A method for detecting a GMDS-BRAF fusion geneencoding the fusion protein in a sample obtained from a subject, usingthe kit according to claim
 9. 14. A method for detecting a GMDS-BRAFfusion gene encoding the fusion protein in a sample obtained from asubject, using the kit according to claim
 10. 15. A method for screeningfor a substance that inhibits the activity and/or expression of apolypeptide comprising the steps of: (1) contacting the polypeptide or acell expressing the polypeptide with a test substance; (2) analyzingwhether or not to inhibit the activity and/or expression of thepolypeptide; and (3) selecting the substance that inhibits the activityand/or expression of the polypeptide, wherein the polypeptide selectedfrom the group consisting of the following polypeptides (a) to (d): (a)a polypeptide consisting of the amino acid sequence represented by SEQID NO: 4 (GMDS-BRAF); (b) a polypeptide comprising the amino acidsequence represented by SEQ ID NO: 4, and having tumorigenicity; (c) apolypeptide comprising an amino acid sequence with 80% or higheridentity to the amino acid sequence represented by SEQ ID NO: 4, andhaving tumorigenicity; and (d) a polypeptide comprising an amino acidsequence derived from the amino acid sequence represented by SEQ ID NO:4 by the deletion, substitution, and/or insertion of one or severalamino acids, and having tumorigenicity.
 16. The screening methodaccording to claim 15, wherein the substance that inhibits the activityand/or expression of the polypeptide is a therapeutic agent for aGMDS-BRAF fusion-positive cancer.